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padding:8px; | padding:8px; | ||
} | } | ||
+ | |||
+ | .kyoto-calender table{ | ||
+ | border-collapse: collapse; | ||
+ | margin: 10px; | ||
+ | margin-right: 4px; | ||
+ | float: left; | ||
+ | } | ||
+ | .kyoto-calendar th{ | ||
+ | width: 24px; | ||
+ | border: 1px solid; | ||
+ | padding: 3px; | ||
+ | background-color: #D6D6D6; | ||
+ | } | ||
+ | |||
+ | .kyoto-calendar td { | ||
+ | width: 24px; | ||
+ | height: 24px; | ||
+ | border: 1px solid; | ||
+ | padding: 0px; | ||
+ | text-align: center; | ||
+ | } | ||
+ | |||
+ | .kyoto-calendar a { | ||
+ | width: 100%; | ||
+ | height: 100%; | ||
+ | display: block; | ||
+ | padding-bottom: 2px; | ||
+ | text-align: center; | ||
+ | background-color: #f8f8ff; | ||
+ | } | ||
+ | |||
+ | .kyoto-calendar a:hover{ | ||
+ | background-color: #ffff00; | ||
+ | } | ||
+ | |||
</style> | </style> | ||
Line 169: | Line 204: | ||
<div id="contents"> | <div id="contents"> | ||
+ | <div class="kyoto-calendar"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=7>August</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>3</td> | ||
+ | <td>4</td> | ||
+ | <td>5</td> | ||
+ | <td>6</td> | ||
+ | <td>7</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>12</td> | ||
+ | <td>13</td> | ||
+ | <td><a href="#0814">14</a></td> | ||
+ | <td><a href="#0815">15</a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="#0816">16</a></td> | ||
+ | <td><a href="#0817">17</a></td> | ||
+ | <td><a href="#0818">18</a></td> | ||
+ | <td><a href="#0819">19</a></td> | ||
+ | <td><a href="#0820">20</a></td> | ||
+ | <td><a href="#0821">21</a></td> | ||
+ | <td>22</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>23</td> | ||
+ | <td><a href="#0824">24</a></td> | ||
+ | <td><a href="#0825">25</a></td> | ||
+ | <td><a href="#0826">26</a></td> | ||
+ | <td><a href="#0827">27</a></td> | ||
+ | <td>28</td> | ||
+ | <td>29</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>30</td> | ||
+ | <td>31</td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=7>September</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>3</td> | ||
+ | <td>4</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>7</td> | ||
+ | <td><a href="#0908">8</a></td> | ||
+ | <td><a href="#0909">9</a></td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>12</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <td><a href="#0914">14</a></td> | ||
+ | <td><a href="#0915">15</a></td> | ||
+ | <td><a href="#0916">16</a></td> | ||
+ | <td><a href="#0917">17</a></td> | ||
+ | <td>18</td> | ||
+ | <td>19</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>20</td> | ||
+ | <td>21</td> | ||
+ | <td>22</td> | ||
+ | <td>23</td> | ||
+ | <td>24</td> | ||
+ | <td>25</td> | ||
+ | <td>26</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>27</td> | ||
+ | <td>28</td> | ||
+ | <td>29</td> | ||
+ | <td>30</td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0814" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>RFP 0.5ng/µl</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>RFP 5ng/µl</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>RFP 10ng/µl</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>RFP 20ng/µl</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>RFP 50ng/µl</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0814" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>10/1 PQ(3)</td><td>about 23.5</td></tr> | ||
+ | <tr><td>2</td><td>1kb ladder</td><td>6</td></tr> | ||
+ | <tr><td>3</td><td>10/1 PQ(3)</td><td>about 23.5</td></tr> | ||
+ | <tr><td>4</td><td>10/1 PQ(3)</td><td>about 24</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/7a/T--Kyoto--8_14_No1_Gel_Extraction_Notsu_09_19_12_14_09No.20814.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">Total volume of samples of lane 1~3 was 71.5µl and it was divided into three.</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Egashira --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0814" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>3C5</td><td>10µl</td><td>DH5α 20µl</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>P+</td><td>10µl</td><td>DH5α 20µl</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>P-</td><td>10µl</td><td>DH5α 20µl</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>RBS</td><td>10µl</td><td>DH5α 20µl</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0814" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Iguchi, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/13 transformation RFP 20ng/µl colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony4</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony5</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony6</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony7</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony8</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony9</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony10</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/13 transformation part RFP colony11</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0815" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>RFP1 /2µl</td><td>82.5</td><td>1.40</td><td>1.22</td></tr> | ||
+ | <tr><td>RFP2 /4µl</td><td>142.5</td><td>1.69</td><td>2.05</td></tr> | ||
+ | <tr><td>RFP3 /4µl</td><td>50</td><td>1.55</td><td>1.71</td></tr> | ||
+ | <tr><td>RFP4 /4µl</td><td>57.5</td><td>1.64</td><td>1.88</td></tr> | ||
+ | <tr><td>RFP6 /4µl</td><td>98.75</td><td>1.71</td><td>1.40</td></tr> | ||
+ | <tr><td>RFP(20) /4µl</td><td>76.25</td><td>1.67</td><td>1.32</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Egashira --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0815" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Notsu, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 3µl</td></tr> | ||
+ | <tr><td>2</td><td>RFP1 10µl</td></tr> | ||
+ | <tr><td>3</td><td>RFP2 10µl</td></tr> | ||
+ | <tr><td>4</td><td>RFP3 10µl</td></tr> | ||
+ | <tr><td>5</td><td>RFP4 10µl</td></tr> | ||
+ | <tr><td>6</td><td>RFP6 10µl</td></tr> | ||
+ | <tr><td>7</td><td>RFP(20) 10µl</td></tr> | ||
+ | <tr><td>8</td><td>PCR1 5µl</td></tr> | ||
+ | <tr><td>9</td><td>PCR2 5µl</td></tr> | ||
+ | <tr><td>10</td><td>PCR3 5µl</td></tr> | ||
+ | <tr><td>11</td><td>PCR4 5µl</td></tr> | ||
+ | <tr><td>12</td><td> PCR5 5 µl</td></tr> | ||
+ | <tr><td>13</td><td>1kb ladder 3µl</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/5e/T--Kyoto--8_15_No6_Electrophoresis_Egashira_02_08_02_44_07.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Egashira --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0815" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Notsu, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/15Miniprep PSB1C3-RFP1</td><td>24</td><td>1</td><td>0</td><td>0</td><td>1</td><td>5(H)</td><td>0</td><td>19</td><td>50</td></tr> | ||
+ | <tr><td>8/15Miniprep PSB1C3-RFP2</td><td>14</td><td>1</td><td>0</td><td>0</td><td>1</td><td>5(H)</td><td>0</td><td>29</td><td>50</td></tr> | ||
+ | <tr><td>8/15Miniprep PSB1C3-RFP3</td><td>40</td><td>1</td><td>0</td><td>0</td><td>1</td><td>5(H)</td><td>0</td><td>3</td><td>50</td></tr> | ||
+ | <tr><td>8/15Miniprep PSB1C3-RFP4</td><td>35</td><td>1</td><td>0</td><td>0</td><td>1</td><td>5(H)</td><td>0</td><td>8</td><td>50</td></tr> | ||
+ | <tr><td>8/15Miniprep PSB1C3-RFP6</td><td>20</td><td>1</td><td>0</td><td>0</td><td>1</td><td>5(H)</td><td>0</td><td>23</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0815" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/15</h3></span> | ||
+ | <span class="keln_exp"><h4>colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>colony Primers</th></tr> | ||
+ | <tr><td>8/14 transformation pSB1C3-RFP1 VF+VR</td></tr> | ||
+ | <tr><td>8/14 transformation pSB1C3-RFP2 VF+VR</td></tr> | ||
+ | <tr><td>8/14 transformation pSB1C3-RFP3 VF+VR</td></tr> | ||
+ | <tr><td>8/14 transformation pSB1C3-RFP4 VF+VR</td></tr> | ||
+ | <tr><td>8/14 transformation pSB1C3-RFP1 VF+VR</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0815" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>94℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>94℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>55℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>68℃</td><td>1min</td><td>30</td></tr> | ||
+ | <tr><td>Final elongation</td><td>4℃</td><td>7min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0816" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>10</td></tr> | ||
+ | <tr><td>2</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/15 digestion pSB1C3-RFP 1</td><td>55</td></tr> | ||
+ | <tr><td>4</td><td>8/15 digestion pSB1C3-RFP 2</td><td>55</td></tr> | ||
+ | <tr><td>5</td><td>8/15 digestion pSB1C3-RFP 3</td><td>36</td></tr> | ||
+ | <tr><td>6</td><td>8/15 digestion pSB1C3-RFP 4</td><td>36</td></tr> | ||
+ | <tr><td>7</td><td>8/15 digestion pSB1C3-RFP 6</td><td>36</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/77/T--Kyoto--8_16_No10_Gel_Extraction_Notsu_09_26_01_32_24.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0816" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>8/16 Gel Extraction RFP1 /1</td><td></td></tr> | ||
+ | <tr><td>2</td><td>8/16 Gel Extraction RFP2 /1</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/16 Gel Extraction RFP3 /1</td><td></td></tr> | ||
+ | <tr><td>4</td><td>8/16 Gel Extraction RFP4 /1</td><td></td></tr> | ||
+ | <tr><td>5</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>6</td><td>8/16 pSB1C3-vector /1</td><td></td></tr> | ||
+ | <tr><td>7</td><td>8/16 pSB1C3-vector /1</td><td></td></tr> | ||
+ | <tr><td>8</td><td>1kladder</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6f/T--Kyoto--8_16_No11_Electrophoresis_Notsu_09_26_01_55_23.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0816" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu, Kaneko, Watanabe</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector DNA /(µl)</th><th>insert DNA /(µl)</th><th>ligase /(µl)</th><th>10×buffer /(µl)</th><th>MilliQ /(µl)</th><th>Total /(µl)</th></tr> | ||
+ | <tr><td>pSB1C3 EPcut /1</td><td>RFP /4</td><td>0.5</td><td>1</td><td>3</td><td>9.5</td></tr> | ||
+ | <tr><td>pSB1C3 EPcut /1</td><td>RFP /2</td><td>0.5</td><td>1</td><td>5</td><td>9.5</td></tr> | ||
+ | <tr><td>pSB1C3 EPcut /1</td><td>RFP /4</td><td>0.5</td><td>1</td><td>3</td><td>9.5</td></tr> | ||
+ | <tr><td>pSB1C3 EPcut /1</td><td>RFP /4</td><td>0.5</td><td>1</td><td>3</td><td>9.5</td></tr> | ||
+ | <tr><td>pSB1C3 EPcut /1</td><td>-</td><td>0.5</td><td>1</td><td>7</td><td>9.5</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0817" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/16 transformation pSB3C5 RFP1</td><td>LB(CP) /6</td></tr> | ||
+ | <tr><td>8/16 transformation pSB3C5 RFP2</td><td>LB(CP) /6</td></tr> | ||
+ | <tr><td>8/16 transformation pSB3C5 RFP1</td><td>LB(CP) /6</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0817" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>pSB1C3 P+</td><td>2</td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>pSB1C3 P-</td><td>2</td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>pSB1C3 RBS</td><td>2</td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>pSB3C5 RFP</td><td>2</td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>8/16 ligation pSB1C3 RFP1</td><td>2</td><td>DH5α</td><td>LB</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0817" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder /3</td><td></td></tr> | ||
+ | <tr><td>2</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>3</td><td>pSB1C3-RFP(ligate)1 /2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>pSB1C3-RFP(ligate)2 /2</td><td></td></tr> | ||
+ | <tr><td>5</td><td>pSB1C3-RFP(ligate)3 /2</td><td></td></tr> | ||
+ | <tr><td>6</td><td>pSB1C3-RFP(ligate)4 /3</td><td></td></tr> | ||
+ | <tr><td>7</td><td>Vector (Ctrl) /5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c7/T--Kyoto--8_17_No14_Electrophoresis_Notsu_09_29_12_26_11.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0818" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>INP [1] BBa_K811005</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>INP [2] BBa_K811005</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>INP [3] BBa_K811005</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv04 [1] BBa_K875004</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv04 [2] BBa_K875004</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv04 [3] BBa_K875004</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv06 [1] BBa__K875006</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv06 [2] BBa_K875006</td><td>LB(CP) / 6ml</td></tr> | ||
+ | <tr><td>scfv06 [3] BBa_K875006</td><td>LB(CP) / 6ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0818" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/17 Liquid Culture 3C5 [1] / 5µl</td><td>77</td><td>1.69</td><td>1.45</td></tr> | ||
+ | <tr><td>8/17 Liquid Culture 3C5 [2] / 2µl</td><td>40</td><td>1.67</td><td>1.17</td></tr> | ||
+ | <tr><td>8/17 Liquid Culture 3C5 [3] / 2µl</td><td>15</td><td>3.75</td><td>3.70</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0818" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>[1] psb3c5(8/18 miniprep) /4.2</td><td>3807</td></tr> | ||
+ | <tr><td>3</td><td>[2] psb3c5(8/18 miniprep) /4.2</td><td>3807</td></tr> | ||
+ | <tr><td>4</td><td>[1'] psb3c5(8/18 miniprep) /4.2</td><td>3807</td></tr> | ||
+ | <tr><td>5</td><td>[2'] psb3c5(8/18 miniprep) /4.2</td><td>3807</td></tr> | ||
+ | <tr><td>6</td><td>[3'] psb3c5(8/18 miniprep) /4.2</td><td>3807</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--8_18_No18_Electrophoresis_Kaneko_05_20_04_12_53.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img18]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0818" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td></td><td>psb3c5(8/18miniprep) [1'], [2'], [3'] mixed / 50</td><td>1.0</td><td></td><td></td><td>1.0</td><td>5.6</td><td></td><td></td><td>56.6</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0819" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Iguchi, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>BBa_ J61002 p+</td><td>2</td><td>DH5a /20</td><td>LB(Amp)</td></tr> | ||
+ | <tr><td>BBa_J61002 p-</td><td>2</td><td>DH5a /20</td><td>LB(Amp)</td></tr> | ||
+ | <tr><td>BBa_1A2 RBS</td><td>2</td><td>DH5a /20</td><td>LB(Amp)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Failed due to deactivation of Amp</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0819" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/19</h3></span> | ||
+ | <span class="keln_exp"><h4>MutationPCR</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates /µl</th><th>Primer /µl</th><th>Primer STAR Max Premix /µl</th><th>MilliQ /µl</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>scfv plasmid /0.1</td><td>m.scfv FW /1, m.scfvRV/1</td><td>25</td><td>22.9</td><td>50</td></tr> | ||
+ | <tr><td>scfv plasmid (nega) /0.1</td><td>0</td><td>5</td><td>4.9</td><td>10</td></tr> | ||
+ | <tr><td>(nega) /0</td><td>m.scfv FW/0.2, m.scfvRV/0.2</td><td>5</td><td>4.6</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">---30cycle---<br>98℃ 10''<br>55℃ 15''<br>72℃ 20''<br>----------------<br>4℃ holding</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0819" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/2</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>scfv plasmid +primer(m.scfv FW, m.scfv RV) mutation PCR product /5</td><td>3600</td></tr> | ||
+ | <tr><td>3</td><td>scfv plasmid mutation PCR product /5</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>primer(m.scfv FW, m.scfv RV) mutation PCR product /5</td><td>-</td></tr> | ||
+ | <tr><td>5</td><td>psb3c5 EPcut product /5</td><td>2600(backborn), 1200(inseert<RFP>)</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/64/T--Kyoto--8_19_No22_Electrophoresis_Kaneko_05_20_04_27_06.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img22]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0819" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Concentration</h4></span> | ||
+ | <span class="keln_researcher">Li, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>OD600</th><th>Bacterial cells /ml</th></tr> | ||
+ | <tr><td>INP [1] K811005</td><td>0.643</td><td>5.14x10^8</td></tr> | ||
+ | <tr><td>scfv06 K875006</td><td>0.634</td><td>5.07x10^8</td></tr> | ||
+ | <tr><td>scfv04 K875004</td><td>0.618</td><td>4.94x10^8</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">1 OD 600 equals to 8x10^8 cells /ml</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0819" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Li, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>scfv 04[1] K875004 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>scfv 04[2] K875004 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>scfv 04[3] K875004 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>scfv 06[1] K875006 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>scfv 06[2] K875006 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>scfv 06[3] K875006 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>INP [1] K811005 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>INP [2] K811005 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | <tr><td>INP [3] K811005 /3µl</td><td>56</td><td>1.63</td><td>1.45</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">There was a problem with an instrument</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0820" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/19 mutation PCR scfv plasmid (nega)</td><td>1</td><td>DH5a/ 20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">8/19 mutation PCR:<br>1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0820" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/19 mutation PCR scfv plasmid</td><td>1 [note:1]</td><td>DH5a/20</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>8/19 mutation PCR scfv plasmid (nega)</td><td>1 [note:2]</td><td>DH5a/20</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>BBa_J61002 p+</td><td>1</td><td>DH5a/20</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>BBa_J61002 p-</td><td>1</td><td>DH5a/20</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>BBa_1A2 RBS</td><td>1</td><td>DH5a/20</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">[note:1]<br>8/19 mutation PCR:<br>1µl extraction of scfv plasmid (nega)/50µl was folded 300 times.<br>[note:2]<br>8/19 mutation PCR:<br>1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0820" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /1.5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/19 miniprep scfv04 [1]</td><td></td></tr> | ||
+ | <tr><td>3</td><td>9/19 miniprep scfv04 [2]</td><td></td></tr> | ||
+ | <tr><td>4</td><td>9/19 miniprep scfv04 [3]</td><td></td></tr> | ||
+ | <tr><td>5</td><td>9/19 miniprep scfv06 [1]</td><td></td></tr> | ||
+ | <tr><td>6</td><td>9/19 miniprep scfv06 [2]</td><td></td></tr> | ||
+ | <tr><td>7</td><td>9/19 miniprep scfv06 [3]</td><td></td></tr> | ||
+ | <tr><td>8</td><td>9/19 miniprep INP [1]</td><td></td></tr> | ||
+ | <tr><td>9</td><td>9/19 miniprep INP [2]</td><td></td></tr> | ||
+ | <tr><td>10</td><td>9/19 miniprep INP [3]</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/53/T--Kyoto--8_20_No26_Electrophoresis_Kaneko_05_20_04_44_45.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img26]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Colony</th><th>Primers</th></tr> | ||
+ | <tr><td>8/20 transformation RBS</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p+</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p-</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 dilution INP</td><td>VF+VR</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre</td><td>94℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>De</td><td>94℃</td><td>30 sec</td><td>30</td></tr> | ||
+ | <tr><td>An</td><td>55℃</td><td>30 sec</td><td>30</td></tr> | ||
+ | <tr><td>Ex</td><td>68℃</td><td>1min</td><td>30</td></tr> | ||
+ | <tr><td>Final E</td><td>4℃</td><td>7min</td><td>1</td></tr> | ||
+ | <tr><td>Final Hold</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Autoclaved distrilled water /23µl<br>2xQuick taq /25µl<br>10 pmol/µl primer (VF) /1µl<br>10 pmol/µl primer (VR) /1µl<br>Template DNA /0µl<br>Total 50µl</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /2</td><td></td></tr> | ||
+ | <tr><td>2</td><td>8/21 colony PCR RBS /all</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/21 colony PCR p+ /all</td><td></td></tr> | ||
+ | <tr><td>4</td><td>8/21 colony PCR p- /all</td><td></td></tr> | ||
+ | <tr><td>5</td><td>8/21 colony PCR INP /all</td><td></td></tr> | ||
+ | <tr><td>6</td><td>8/21 colony PCR Nega /all</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/04/T--Kyoto--8_21_No28_Electrophoresis_Kaneko_05_20_04_54_01%2C1.jpg"><img src="https://static.igem.org/mediawiki/2016/b/b5/T--Kyoto--8_21_No28_Electrophoresis_Kaneko_05_20_04_54_01%2C2.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img29-1]<br>weak band except INP -> retry<br>colony PCR and Electrophoresis were conducted under the same condition.<br>[img29-2]<br>RBS and INP success.<br>P+ and P- failed -> retry.</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>70℃</td><td>15sec</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>scfv 8/20 transformation</td><td>cscfv.f /0.75</td><td>cscfv.r /0.75</td><td>2x KAPA Hifi /12.5</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>- (nega)</td><td>cscfv.f /0.3</td><td>cscfv.f /0.3</td><td>2x KAPA Hifi /5</td><td>4.4</td><td></td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Colony</th><th>Primers</th></tr> | ||
+ | <tr><td>8/20 transformation p+ [1]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p+ [2]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p+ [3]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p- [1]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p- [2]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/20 transformation p- [3]</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/18 parts p+</td><td>VF+VR</td></tr> | ||
+ | <tr><td>8/18 parts p-</td><td>VF+VR</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>pre</td><td>94℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>de</td><td>94℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>an</td><td>55℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>ex</td><td>68℃</td><td>1min</td><td>30</td></tr> | ||
+ | <tr><td>F.elong</td><td>4℃</td><td>7min</td><td>1</td></tr> | ||
+ | <tr><td>F.hold</td><td>4℃</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>strain</td><td>Medium/ml</td></tr> | ||
+ | <tr><td>8/20 transformation RBS</td><td>PG(AMP) /6</td></tr> | ||
+ | <tr><td>8/20 dilution INP7</td><td>PG(CP) /6</td></tr> | ||
+ | <tr><td>8/17 transformation 3c5, RFP</td><td>PG(CP) /6</td></tr> | ||
+ | <tr><td>8/20 transformation p+ [1]</td><td>PG(AMP) /6</td></tr> | ||
+ | <tr><td>8/20 transformation p- [1]</td><td>PG(AMP) /6</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /1.5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>8/21 cloning PCR scfv[1] /2</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/21 cloning PCR scfv[2] /2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>nega /2</td><td></td></tr> | ||
+ | <tr><td>5</td><td>8/21 colony PCR p+ [1] /2</td><td></td></tr> | ||
+ | <tr><td>6</td><td>8/21 colony PCR p+ [2] /2</td><td></td></tr> | ||
+ | <tr><td>7</td><td>8/21 colony PCR p+ [3] /2</td><td></td></tr> | ||
+ | <tr><td>8</td><td>8/21 colony PCR p- [1] /2</td><td></td></tr> | ||
+ | <tr><td>9</td><td>8/21 colony PCR p- [2] /2</td><td></td></tr> | ||
+ | <tr><td>10</td><td>8/21 colony PCR p- [3] /2</td><td></td></tr> | ||
+ | <tr><td>11</td><td>8/21 colony PCR parts p+ /2</td><td></td></tr> | ||
+ | <tr><td>12</td><td>8/21 colony PCR parts p- /2</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/9d/T--Kyoto--8_21_No34_Electrophoresis_Kaneko_05_20_05_13_01.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img35]<br>2, 3, 4 cloning PCR failed.<br>1 forgot to put ladder<br>5, 7, 8, 9 strong band<br>6 weak<br>10 failed<br>P+[1] and p-[1] -> liquid culture</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample/µl</th><th>MilliQ (µl)</th><th>Buffer /µl</th><th>Ligase(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>8/19 mutation PCR scfv /5</td><td>3.5</td><td>1</td><td>0.5</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/20 transformation m.scfv colonyA /6</td><td>PG(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/20 Liquid culture RBS</td><td>124.5</td><td>1.65</td><td>1.02</td></tr> | ||
+ | <tr><td>8/20 Liquid culture INP</td><td>147.8</td><td>1.66</td><td>0.98</td></tr> | ||
+ | <tr><td>8/20 Liquid culture 3c5.RFP</td><td>234.8</td><td>1.79</td><td>1.62</td></tr> | ||
+ | <tr><td>8/20 Liquid culture P+[1]</td><td>40.3</td><td>1.78</td><td>1.60</td></tr> | ||
+ | <tr><td>8/20 Liquid culture P-[1]</td><td>56.7</td><td>1.80</td><td>2.05</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino, Notsu, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/24 LIquid Culture m.scfv colonyA</td><td>-</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>8/24 LIquid Culture m.scfv colonyB</td><td>-</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /1.5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>8/24 miniprep p+(J61002) /2</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/24 miniprep p-(J61002) /2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>8/24 miniprep RFP (3c5) /2</td><td></td></tr> | ||
+ | <tr><td>5</td><td>8/24 miniprep INP (1c3) /2</td><td></td></tr> | ||
+ | <tr><td>6</td><td>8/24 miniprep RBS (1A2) /2</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/fd/T--Kyoto--8_25_No38_Electrophoresis_Kaneko_05_20_05_22_35.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img38]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td></td><td>p+/24.8</td><td></td><td></td><td>1</td><td>1</td><td>10xH /4</td><td></td><td>9.2</td><td>40</td></tr> | ||
+ | <tr><td></td><td>IINP /6.8</td><td></td><td>1</td><td></td><td>1</td><td>10xM /4</td><td></td><td>27.2</td><td>40</td></tr> | ||
+ | <tr><td></td><td>p- /17.6</td><td></td><td></td><td>1</td><td>1</td><td>10xH /4</td><td></td><td>16.4</td><td>40</td></tr> | ||
+ | <tr><td></td><td>RBS /8.0</td><td></td><td></td><td>1</td><td>1</td><td>10xH /4</td><td></td><td>26</td><td>40</td></tr> | ||
+ | <tr><td></td><td>RFP /4.3</td><td>1</td><td></td><td></td><td>1</td><td>10xH /4</td><td></td><td>29.7</td><td>40</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>6</td></tr> | ||
+ | <tr><td>2, 3</td><td>8/25 digestion p+</td><td>24 each</td></tr> | ||
+ | <tr><td>4, 5</td><td>8/25 digestion p- </td><td>24 each</td></tr> | ||
+ | <tr><td>6, 7</td><td>8/25 digestion RFP </td><td>24 each</td></tr> | ||
+ | <tr><td>8, 9</td><td>8/25 digestion INP </td><td>24 each</td></tr> | ||
+ | <tr><td>10, 11</td><td>8/25 digestion RBS </td><td>24 each</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/77/T--Kyoto--8_25_No40_Gel_Extraction_Kaneko_05_20_05_28_41.JPG"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img40]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Actvation check of Xba and Pst</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>INP(µl)</th><th>Xba(µl)</th><th>Pst(µl)</th><th>10xM(µl)</th><th>BSA(µl)</th><th>MilliQ(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>0.68</td><td>0.1</td><td>0.1</td><td>1</td><td>0.2</td><td>7.92</td><td>10</td></tr> | ||
+ | <tr><td>0.68</td><td>0.1</td><td>-</td><td>1</td><td>0.2</td><td>8.02</td><td>10</td></tr> | ||
+ | <tr><td>0.68</td><td>-</td><td>0.1</td><td>1</td><td>0.2</td><td>8.02</td><td>10</td></tr> | ||
+ | <tr><td>0.68</td><td>-</td><td>-</td><td>1</td><td>0.2</td><td>8.12</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /3</td><td></td></tr> | ||
+ | <tr><td>2</td><td>[1] (inp xp cut) /10</td><td></td></tr> | ||
+ | <tr><td>3</td><td>[2] (inp x cut) /10</td><td></td></tr> | ||
+ | <tr><td>4</td><td>[3] (inp p cut) /10</td><td></td></tr> | ||
+ | <tr><td>5</td><td>[4] (inp no cut) /10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/46/T--Kyoto--8_25_No41_2_Electrophoresis_Kaneko_05_20_05_34_47.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img42]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td></td><td>8/24 miniprep INP 1c3 /0.68</td><td>-</td><td>1.0</td><td></td><td></td><td></td><td>0.2</td><td>7.12</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/15 miniprep RFP psb1c3 /1.3</td><td>-</td><td>1.0</td><td></td><td></td><td></td><td>0.2</td><td>6.5</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/24 miniprep INP 1c3 /0.68</td><td>0.1</td><td>1.0</td><td></td><td></td><td></td><td>0.2</td><td>7.02</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/15 miniprep RFP psb1c3 /1.3</td><td>0.1</td><td>1.0</td><td></td><td></td><td></td><td>0.2</td><td>6.4</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/24 miniprep INP 1c3/ 0.68</td><td>0.1</td><td>-</td><td></td><td></td><td></td><td>0.2</td><td>8.02</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/15 miniprep RFP psb1c3 /1.3</td><td>0.1</td><td>-</td><td></td><td></td><td></td><td>0.2</td><td>7.4</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/24 miniprep INP 1c3 /0.68</td><td>-</td><td>-</td><td></td><td></td><td></td><td>0.2</td><td>8.12</td><td>10</td></tr> | ||
+ | <tr><td></td><td>8/15 miniprep RFP psb1c3 /1.3</td><td>-</td><td>-</td><td></td><td></td><td></td><td>0.2</td><td>7.5</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>scfv from MK</td><td>c.scfv fw/0.75</td><td>c.scfv rv/0.75</td><td>2x KAPA Hifi /12.5</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>none</td><td>c.scfv fw/0.75</td><td>c.scfv rv/0.75</td><td>2x KAPA Hifi /5</td><td>4.4</td><td></td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Activation Check of Xba</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/2</td><td></td></tr> | ||
+ | <tr><td>2</td><td>[8] RFP/10</td><td></td></tr> | ||
+ | <tr><td>3</td><td>[6] RFP EcoRI/10</td><td></td></tr> | ||
+ | <tr><td>4</td><td>[2] RFP XbaI/10</td><td></td></tr> | ||
+ | <tr><td>5</td><td>[4] RFP EcoRI, XbaI /10</td><td></td></tr> | ||
+ | <tr><td>6</td><td>[7] INP /10</td><td></td></tr> | ||
+ | <tr><td>7</td><td>[5] INP EcoRI /10</td><td></td></tr> | ||
+ | <tr><td>8</td><td>[1] INP XbaI /10</td><td></td></tr> | ||
+ | <tr><td>9</td><td>[3] INP EcoRI, XbaI/10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/17/T--Kyoto--8_26_No45_Electrophoresis_Kaneko_05_20_05_42_54.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">[img45-1]<br>[img45-2]</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0827" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>2</td><td>1kb ladder</td><td>2</td></tr> | ||
+ | <tr><td>3</td><td>8/25 PCR scfv</td><td>5</td></tr> | ||
+ | <tr><td>4</td><td>8/25 PCR none</td><td>5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/fc/T--Kyoto--8_27_No46_Electrophoresis_Iguchi_10_17_06_08_50.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0827" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/27</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>s.BAN</td><td>BAN.Fw/ 0.75</td><td>BAN.Rv/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>s.BAN</td><td>BAN.Fw/ 0.75</td><td>BAN.Rv/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>s.BAN</td><td>BAN.Fw/ 0.75</td><td>BAN.Histag/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>scfv</td><td>scfv.Fw/ 0.75</td><td>scfv.Rv/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/24 miniprep INP</td><td>INP.Fw/ 0.75</td><td>INP.Rv/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/24 miniprep INP</td><td>INP.Fw/ 0.75</td><td>INP.Rv.Histag/ 0.75</td><td></td><td>11</td><td>2xKAPA HiFi/ 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0827" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/27</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98</td><td>20sec</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>65</td><td>15sec</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>72</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>Final Elogation</td><td>72</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kaneko --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td></td><td>[1] 8/15 miniprep 1c3 RFP /14</td><td>0.5</td><td></td><td>0.5</td><td>-</td><td>10xH /3</td><td></td><td>12</td><td>30</td></tr> | ||
+ | <tr><td></td><td>[2] 8/24 miniprep 3c5 RFP /8.5</td><td>0.5</td><td></td><td>-</td><td>0.5</td><td>10xH /3</td><td></td><td>17.5</td><td>30</td></tr> | ||
+ | <tr><td></td><td>[3] 8/24 miniprep 3c5 RFP /8.5</td><td>0.5</td><td></td><td>0.5</td><td>-</td><td>10xH /3</td><td></td><td>17.5</td><td>30</td></tr> | ||
+ | <tr><td></td><td>[4] 8/24 miniprep BBa_J61002 p- /24</td><td>0.5</td><td></td><td>-</td><td>0.5</td><td>10xH /3</td><td></td><td>2</td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4c/T--Kyoto--9_8_No50_Restriction_Enzyme_Digestion_Kaneko_05_20_05_48_28.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>1</td><td>100bp ladder 9µL</td></tr> | ||
+ | <tr><td>2</td><td>8/24 miniprep p- EPcut 30µL</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Sukegawa, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder 9µL</td></tr> | ||
+ | <tr><td>2</td><td>8/15 miniprep 1C3RFP EScut 30µL</td></tr> | ||
+ | <tr><td>3</td><td>8/24 miniprep 3C5RFP EPcut 30µL</td></tr> | ||
+ | <tr><td>4</td><td>8/24 miniprep 3C5RFP EScut 30µL</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/24 miniprep INP</td><td>INPfw/0.75</td><td>INPRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>8/24 miniprep INP</td><td>INPfw/0.75</td><td>INPHisRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>BAN</td><td>BANFw/0/75</td><td>BANRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>BAN</td><td>BANFw/0/75</td><td>BANHisRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>BAN </td><td>BANFw/0/75</td><td>RBSRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>scFv fromMk</td><td>scFvFw/0/75</td><td>scFvRv/0/75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | <tr><td>scFv fromMk</td><td>scFvHisFw/0.75</td><td>scFvRv/0.75</td><td>2×KAPAHiFi</td><td>11</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>1</td><td>95</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>2</td><td>98</td><td>70sec</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>68</td><td>15sec</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>72</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>5</td><td>72</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>6</td><td>10</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------><!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>2</td><td>ladder/6</td></tr> | ||
+ | <tr><td>4</td><td>9/14 PCR1/10</td></tr> | ||
+ | <tr><td>6</td><td>9/14 PCR2/10</td></tr> | ||
+ | <tr><td>8</td><td>9/14 PCR3/10</td></tr> | ||
+ | <tr><td>10</td><td>9/14 PCR4/10</td></tr> | ||
+ | <tr><td>12</td><td>9/14PCR5/10</td></tr> | ||
+ | <tr><td>14</td><td>9/14 PCR6/10</td></tr> | ||
+ | <tr><td>16</td><td>9/14 PCR7/10</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4b/T--Kyoto--9_14_No53_Electrophoresis_Yamamoto_08_23_11_27_01.1.JPG"><img src="https://static.igem.org/mediawiki/2016/7/77/T--Kyoto--9_14_No53_Electrophoresis_Yamamoto_08_23_11_27_01.2.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3sBAN/0.15+6scFvfromMK/0.15</td><td>BANFw/0/75</td><td>scFvRv/0.75</td><td>2×KAPAHiFi/12.5</td><td></td><td></td><td>25</td></tr> | ||
+ | <tr><td>4sBAN/0.15+7scFvfromMK/0.15</td><td>BANFw/0/75</td><td>scFvRv/0.75</td><td>2×KAPAHiFi/12.5</td><td></td><td></td><td>25</td></tr> | ||
+ | <tr><td>1INP8/24miniprep/0.15+5sBAN0.15+scFvfromMK/0.15</td><td>BANFw/0/75</td><td>scFvRv/0.75</td><td>2×KAPAHiFi/12.5</td><td></td><td></td><td>25</td></tr> | ||
+ | <tr><td>2INP8/24miniprep/0.15</td><td>BANFw/0/75</td><td>scFvRv/0.75</td><td>2×KAPAHiFi/12.5</td><td></td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3sBAN+6scFvfromMKPCRproduct</td><td>34</td><td>1</td><td></td><td>1</td><td></td><td>4</td><td></td><td></td><td>40</td></tr> | ||
+ | <tr><td>4sBAN+7scFvfromMK PCRproduct</td><td>34</td><td>1</td><td></td><td>1</td><td></td><td>4</td><td></td><td></td><td>40</td></tr> | ||
+ | <tr><td>1INP8/24miniprep+5sBAN+6scFvfromMKPCRproduct</td><td>16</td><td>1</td><td></td><td>1</td><td></td><td>2</td><td></td><td></td><td>20</td></tr> | ||
+ | <tr><td>2INP8/24miniprep+5sBAN+7scFvfromMkPCRproduct</td><td>34</td><td>1</td><td></td><td>1</td><td></td><td>4</td><td></td><td></td><td>40</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0915" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3sBAN+6scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>3</td><td>3sBAN+6scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>4</td><td>4sBAN+7scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>5</td><td>4sBAN+7scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>6</td><td>1INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>7</td><td>2INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>8</td><td>2INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut</td><td>20</td></tr> | ||
+ | <tr><td>9</td><td>RFP3C5PCRproduct</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a2/T--Kyoto--9_15_No56_Gel_Extraction_Yamamoto_08_23_11_46_18%2C1.jpg"><img src="https://static.igem.org/mediawiki/2016/5/52/T--Kyoto--9_15_No56_Gel_Extraction_Yamamoto_08_23_11_46_18%2C2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (2Column)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name I</th><th>Name II</th></tr> | ||
+ | <tr><td>9/15 transformation BANscFv 3C5 colony1~16</td><td>VF+VR</td></tr> | ||
+ | <tr><td>9/15 transformation INPscFv 3C5 colony1~16</td><td>VF+VR</td></tr> | ||
+ | <tr><td>9/15 transformation BANHisscFv 3C5 colony1~16</td><td>VF+VR</td></tr> | ||
+ | <tr><td>9/15 transformation INPHisscFv 3C5 colony1~16</td><td>VF+VR</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>1</td><td>94</td><td>2</td><td>1</td></tr> | ||
+ | <tr><td>2</td><td>94</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>55</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>68</td><td>1</td><td>25</td></tr> | ||
+ | <tr><td>5</td><td>4</td><td>7</td><td>1</td></tr> | ||
+ | <tr><td>6</td><td>4</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Li, Kaneko, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>BANscFv3C5 colony 9</td><td>10</td></tr> | ||
+ | <tr><td>3</td><td>BANscFv3C5 colony 10</td><td>10</td></tr> | ||
+ | <tr><td>4</td><td>BANscFv3C5 colony 11</td><td>10</td></tr> | ||
+ | <tr><td>5</td><td>BANscFv3C5 colony 12</td><td>10</td></tr> | ||
+ | <tr><td>6</td><td>BANscFv3C5 colony 13</td><td>10</td></tr> | ||
+ | <tr><td>7</td><td>BANscFv3C5 colony 14</td><td>10</td></tr> | ||
+ | <tr><td>8</td><td>BANscFv3C5 colony 15</td><td>10</td></tr> | ||
+ | <tr><td>9</td><td>BANscFv3C5 colony 16</td><td>10</td></tr> | ||
+ | <tr><td>10</td><td>BANHisscFv3C5 colony 9</td><td>10</td></tr> | ||
+ | <tr><td>11</td><td>BANHisscFv3C5 colony 10</td><td>10</td></tr> | ||
+ | <tr><td>12</td><td>BANHisscFv3C5 colony 11</td><td>10</td></tr> | ||
+ | <tr><td>13</td><td>BANHisscFv3C5 colony 12</td><td>10</td></tr> | ||
+ | <tr><td>14</td><td>BANHisscFv3C5 colony 13</td><td>10</td></tr> | ||
+ | <tr><td>15</td><td>BANHisscFv3C5 colony 14</td><td>10</td></tr> | ||
+ | <tr><td>16</td><td>BANHisscFv3C5 colony 15</td><td>10</td></tr> | ||
+ | <tr><td>17</td><td>BANHisscFv3C5 colony 16</td><td>10</td></tr> | ||
+ | <tr><td>18</td><td>BANscFv3C5 colony 1</td><td>10</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.1.JPG"><img src="https://static.igem.org/mediawiki/2016/3/34/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.2.JPG"><img src="https://static.igem.org/mediawiki/2016/a/aa/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.3.JPG"><img src="https://static.igem.org/mediawiki/2016/6/67/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.4.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Li, Kaneko, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>INPscFv3C5 colony 9</td><td>10</td></tr> | ||
+ | <tr><td>3</td><td>INPscFv3C5 colony 10</td><td>10</td></tr> | ||
+ | <tr><td>4</td><td>INPscFv3C5 colony 11</td><td>10</td></tr> | ||
+ | <tr><td>5</td><td>INPscFv3C5 colony 12</td><td>10</td></tr> | ||
+ | <tr><td>6</td><td>INPscFv3C5 colony 13</td><td>10</td></tr> | ||
+ | <tr><td>7</td><td>INPscFv3C5 colony 14</td><td>10</td></tr> | ||
+ | <tr><td>8</td><td>INPscFv3C5 colony 15</td><td>10</td></tr> | ||
+ | <tr><td>9</td><td>INPscFv3C5 colony 16</td><td>10</td></tr> | ||
+ | <tr><td>10</td><td>INPHisscFv3C5 colony 9</td><td>10</td></tr> | ||
+ | <tr><td>11</td><td>INPHisscFv3C5 colony 10</td><td>10</td></tr> | ||
+ | <tr><td>12</td><td>INPHisscFv3C5 colony 11</td><td>10</td></tr> | ||
+ | <tr><td>13</td><td>INPHisscFv3C5 colony 12</td><td>10</td></tr> | ||
+ | <tr><td>14</td><td>INPHisscFv3C5 colony 13</td><td>10</td></tr> | ||
+ | <tr><td>15</td><td>INPHisscFv3C5 colony 14</td><td>10</td></tr> | ||
+ | <tr><td>16</td><td>INPHisscFv3C5 colony 15</td><td>10</td></tr> | ||
+ | <tr><td>17</td><td>INPHisscFv3C5 colony 16</td><td>10</td></tr> | ||
+ | <tr><td>18</td><td>BANscFv3C5 colony 1</td><td>10</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/3d/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C1.jpg"><img src="https://static.igem.org/mediawiki/2016/8/88/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C2.jpg"><img src=""><img src="https://static.igem.org/mediawiki/2016/4/42/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C3.jpg"><img src="https://static.igem.org/mediawiki/2016/6/69/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C4.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>1</td><td>buffer/5</td></tr> | ||
+ | <tr><td>2</td><td>1kb ladder/5</td></tr> | ||
+ | <tr><td>3</td><td>BAN 9 P/4.5</td></tr> | ||
+ | <tr><td>4</td><td>BAN 9 N/4.5</td></tr> | ||
+ | <tr><td>5</td><td>BAN14 P/4.5</td></tr> | ||
+ | <tr><td>6</td><td>BAN14 N/4.5</td></tr> | ||
+ | <tr><td>7</td><td>BANHis14 P/4.5</td></tr> | ||
+ | <tr><td>8</td><td>BANHis14/4.5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--9_17_No62_Electrophoresis_Yamamoto_08_23_12_42_58.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0917" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>none</td><td></td></tr> | ||
+ | <tr><td>3</td><td>BANHis16 p</td><td></td></tr> | ||
+ | <tr><td>4</td><td>BANHis16 n</td><td></td></tr> | ||
+ | <tr><td>5</td><td>INP 12 p</td><td></td></tr> | ||
+ | <tr><td>6</td><td>INP 14 p</td><td></td></tr> | ||
+ | <tr><td>7</td><td>INP 12 n</td><td></td></tr> | ||
+ | <tr><td>8</td><td>INP 14 n</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/9b/T--Kyoto--9_17_No63_Electrophoresis_Iguchi_10_17_06_39_35.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto,Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0917" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Products</th></tr> | ||
+ | <tr><td>1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>1kbladder/5</td></tr> | ||
+ | <tr><td>4</td><td>INPHis9 P/4.5</td></tr> | ||
+ | <tr><td>5</td><td>INPHis9N/4.5</td></tr> | ||
+ | <tr><td>6</td><td>INPHis12P/4.5</td></tr> | ||
+ | <tr><td>7</td><td>INPHis12N/4.5</td></tr> | ||
+ | <tr><td>8</td><td>INP14 N/4.5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b8/T--Kyoto--9_17_No64_Electrophoresis_Yamamoto_08_23_01_05_58.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0917" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>BAN9 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>BAN9 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>BAN14 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>BAN14 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>BANHis14 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>BANHis 14 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>BANHis 16 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>BANHis16 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>INP12 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>INP12 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>INP14 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>INP 14 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>INP 9 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>INP9 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | <tr><td>INPHis12 </td><td>2</td><td>0.5</td><td></td><td></td><td>0.5</td><td>10×H/1</td><td></td><td>6</td><td>10</td></tr> | ||
+ | <tr><td>INPHis12 </td><td>1</td><td>0</td><td></td><td></td><td>0</td><td>10×H/1</td><td></td><td>8</td><td>10</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--9_17_No65_Restriction_Enzyme_Digestion_Yamamoto_08_23_01_13_52.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0917" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/16 liquid culture BANscFv12</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture BANscFv14</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture BANHisscFv12</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture BANHisscFv14</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture INPHisscFv12</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture INPHisscFv14</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture INPHisscFv10</td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>9/16 liquid culture BANscFv</td><td></td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0204" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/4</h3></span> | ||
+ | <span class="keln_exp"><h4>PartsAwaking</h4></span> | ||
+ | <span class="keln_researcher">Kaneko, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Description</th><th>PartName</th><th>Backbone</th><th>Well</th><th>PlateYear</th><th>PlateNumber</th><th>MilliQ(µl)</th></tr> | ||
+ | <tr><td>RFP</td><td>E1010</td><td>pSB1C3</td><td>11N</td><td>2015</td><td>3</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0204" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Iguchi, Notsu, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>E1010</td><td>2</td><td>DH5α</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0205" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/4 transformation E1010 colony1</td><td>VF 6.25µl VR 6.25µl</td><td>12.5</td><td>2×Quick Taq HS DyeMix 25µl</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0205" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/5</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>94℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>94℃</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>51℃</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>68℃</td><td>90sec</td><td>25</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>4℃</td><td>-</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yamamoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0205" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>9/17miniprep BAN9</td><td>2</td><td>DH5α/20µL</td><td></td></tr> | ||
+ | <tr><td>9/17miniprep BANHis14</td><td>2</td><td>DH5α/20µL</td><td></td></tr> | ||
+ | <tr><td>9/17miniprep INPHis12</td><td>2</td><td>DH5α/20µL</td><td></td></tr> | ||
+ | <tr><td>9/17miniprep INPHis9</td><td>2</td><td>DH5α/20µL</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0205" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/4 transformation E1010 colony1</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0205" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/5 colony PCR E1010</td><td>1019</td></tr> | ||
+ | <tr><td>3</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>5</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>7</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>8</td><td>other sample</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/44/T--Kyoto--2_5_No75_Electrophoresis_Notsu_10_01_04_31_28.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0206" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Notsu, Kaneko, Egashira, Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/5 liquid culture E1010(RFP) colony1</td><td>48.0</td><td>1.93</td><td>1.42</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0206" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Kaneko, Egashira, Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/6 miniprep E1010 colony1</td><td>34</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1×M /4</td><td>0</td><td>0</td><td>40</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0207" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kaneko, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>10</td></tr> | ||
+ | <tr><td>2</td><td>E1010 colony1 2/6 XPcut</td><td>40</td></tr> | ||
+ | <tr><td>3</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>other sample</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/84/T--Kyoto--2_7_No78_Gel_Extraction_Notsu_10_01_04_37_25.1.JPG"><img src="https://static.igem.org/mediawiki/2016/6/68/T--Kyoto--2_7_No78_Gel_Extraction_Notsu_10_01_04_37_25.2.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0207" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Kaneko, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>E1010 colony1 2/6 XPcut</td><td>8.2</td><td>1.03</td><td>0.49</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/71/T--Kyoto--2_7_No79_Gel_Extraction%28Measurement%29_Notsu_10_01_04_40_48.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/71/T--Kyoto--2_7_No79_Gel_Extraction%28Measurement%29_Notsu_10_01_04_40_48.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0207" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/5 transformation BAN9 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/5 transformation BAN-HIS14 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/5 transformation INP-HIS12 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/5 transformation BAN9 colony1</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0208" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/4 transformation E1010 colony2 1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/4 transformation E1010 colony2 2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0208" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/7 Liquid culture BAN9</td><td>123.6</td><td>1.87</td><td>2.07</td></tr> | ||
+ | <tr><td>2/7 Liquid culture BAN9</td><td>248.8</td><td>1.83</td><td>1.84</td></tr> | ||
+ | <tr><td>2/7 Liquid culture BAN-HIS14</td><td>268.9</td><td>1.86</td><td>2.15</td></tr> | ||
+ | <tr><td>2/7 Liquid culture BAN-HIS14</td><td>127.9</td><td>1.85</td><td>2.08</td></tr> | ||
+ | <tr><td>2/7 Liquid culture INP-HIS9</td><td>354.3</td><td>1.86</td><td>2.24</td></tr> | ||
+ | <tr><td>2/7 Liquid culture INP-HIS9</td><td>289.4</td><td>1.85</td><td>1.92</td></tr> | ||
+ | <tr><td>2/7 Liquid culture INP-HIS12</td><td>172.3</td><td>1.87</td><td>2.07</td></tr> | ||
+ | <tr><td>2/7 Liquid culture INP-HIS12</td><td>286.5</td><td>1.86</td><td>2.20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0208" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/8 miniprep BAN9 /5</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2/8 miniprep BAN9 /5</td><td></td></tr> | ||
+ | <tr><td>4</td><td>2/8 miniprep BAN-HIS14 /5</td><td></td></tr> | ||
+ | <tr><td>5</td><td>2/8 miniprep BAN-HIS14 /5</td><td></td></tr> | ||
+ | <tr><td>6</td><td>2/8 miniprep INP-HIS9 /5</td><td></td></tr> | ||
+ | <tr><td>7</td><td>2/8 miniprep INP-HIS9 /5</td><td></td></tr> | ||
+ | <tr><td>8</td><td>2/8 miniprep INP-HIS14 /5</td><td></td></tr> | ||
+ | <tr><td>9</td><td>2/8 miniprep INP-HIS9 /5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/0b/T--Kyoto--2_8_No85_Electrophoresis_Notsu_09_29_01_38_11.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0209" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Matsumoto, Notsu, Egashira, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/8 liquid culture E1010 colony2 1and 2</td><td>58.5</td><td>1.89</td><td>2.16</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0209" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 liquid culture E1010 colony2 </td><td>30</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×M /4</td><td>2</td><td>2</td><td>40</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0209" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 liquid culture E1010 colony2 </td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10×M /2</td><td>1</td><td>12</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Takimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0210" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/9 Negative Control E1010 colony2 /10</td><td>2776</td></tr> | ||
+ | <tr><td>3</td><td>2/9 Digestion E1010 colony2 p+x /40</td><td>732</td></tr> | ||
+ | <tr><td>4</td><td>2/9 Negative Control E0040 colony2 /20</td><td>2799</td></tr> | ||
+ | <tr><td>5</td><td>2/9 Digestion E0040 colony2 p+x /40</td><td>746</td></tr> | ||
+ | <tr><td>6</td><td>2/9 Negative Control K592009 colony2 /20</td><td>2739</td></tr> | ||
+ | <tr><td>7</td><td>2/9 Digestion K592009 colony2 p+x /40</td><td>695</td></tr> | ||
+ | <tr><td>8</td><td>2/9 Negative Control E0022 colony2 /20</td><td>2832</td></tr> | ||
+ | <tr><td>9</td><td>2/9 Digestion E0022 colony2 p+x /40</td><td>788</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Takimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0210" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2/9 NC J23105 colony2 /20</td><td>2105</td></tr> | ||
+ | <tr><td>4</td><td>2/9 Digestion J23105 colony2 E+S /40</td><td>58</td></tr> | ||
+ | <tr><td>5</td><td>2/9 NC J23100 colony2 /20</td><td>2105</td></tr> | ||
+ | <tr><td>6</td><td>2/9 Digestion J23100 colony2 E+S /40</td><td>58</td></tr> | ||
+ | <tr><td>7</td><td>2/9 NC B0034 colony3 /20</td><td>2091</td></tr> | ||
+ | <tr><td>8</td><td>2/9 Digestion B0034 colony3 P+S /40</td><td>2080</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.1.JPG"><img src="https://static.igem.org/mediawiki/2016/4/45/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.2.JPG"><img src="https://static.igem.org/mediawiki/2016/7/73/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.3.JPG"><img src="https://static.igem.org/mediawiki/2016/b/b0/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.4.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Takimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0210" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Egashira, Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>E1010</td><td>9.4</td><td>2.13</td><td>0.61</td></tr> | ||
+ | <tr><td>K592009</td><td>5.2</td><td>1.74</td><td>0.29</td></tr> | ||
+ | <tr><td>E0040</td><td>15.0</td><td>1.94</td><td>0.38</td></tr> | ||
+ | <tr><td>E0022</td><td>8.5</td><td>1.04</td><td>0.86</td></tr> | ||
+ | <tr><td>B0034</td><td>21.2</td><td>1.84</td><td>1.06</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Ideal MIniprep effficiency of E1010 would be 18.5ng/microL (presuming 100% Restriction Enzyme Digestion efficiency and 80 % Gel Extraction efficiency).</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>PartsAwaking</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Description</th><th>PartName</th><th>Backbone</th><th>Well</th><th>PlateYear</th><th>PlateNumber</th><th>MilliQ(µl)</th></tr> | ||
+ | <tr><td>sfGFP-CBDcex</td><td>K1321342</td><td>pSB1C3</td><td>18E</td><td>2015</td><td>5</td><td>10</td></tr> | ||
+ | <tr><td>sfGFP-CBDclos</td><td>K1321341</td><td>pSB1C3</td><td>18C</td><td>2015</td><td>5</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/12 parts awakening sfGFP-CBDcex</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/12 parts awakening sfGFP-CBDclos</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 miniprep BAN-His14 4 /0.1</td><td>VF /0.75</td><td>m.scFv.Rv /0.75</td><td>-</td><td>10.9</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep BAN-His14 4 /0.1</td><td>VR /0.75</td><td>m.scFv.Fw /0.75</td><td>-</td><td>10.9</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>2/12 PCR VF.m.scFv.Rv</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>2/12 PCR VR.m.Fw</td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/0a/T--Kyoto--2_12_No101_Electrophoresis_Notsu_10_01_07_30_03%2C1.jpg"> <img src="https://static.igem.org/mediawiki/2016/c/c8/T--Kyoto--2_12_No101_Electrophoresis_Notsu_10_01_07_30_03%2C2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>PartsAwaking</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Description</th><th>PartName</th><th>Backbone</th><th>Well</th><th>PlateYear</th><th>PlateNumber</th><th>MilliQ(µl)</th></tr> | ||
+ | <tr><td>Promoter</td><td>J23114</td><td>J61002</td><td>19J</td><td>2015</td><td>4</td><td>10</td></tr> | ||
+ | <tr><td>Promoter</td><td>J23108</td><td>pSB1C3</td><td>4C</td><td>2015</td><td>4</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/12 Parts awakening J23114</td><td>1</td><td>DH5α /20</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>2/12 Parts awakening J23108</td><td>1</td><td>DH5α /20</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0212" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/12 PCR VF.m.scFv.Rv </td><td>18.8</td><td>1.95</td><td>1.52</td></tr> | ||
+ | <tr><td>2/12 PCR VR.m.scFv.Fw </td><td>28.6</td><td>2.08</td><td>0.07</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDcex colony1</td><td>VF /0.75 VR /0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDcex colony2</td><td>VF /0.75 VR /0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDclos colony1</td><td>VF /0.75 VR /0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDclos colony2</td><td>VF /0.75 VR /0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/12 transformation sfGFP-CBDcex colony1</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2/12 transformation sfGFP-CBDcex colony2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>2/12 transformation sfGFP-CBDclos colony1</td><td></td></tr> | ||
+ | <tr><td>5</td><td>2/12 transformation sfGFP-CBDclos colony2</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/32/T--Kyoto--2_15_No3_Electrophoresis_Notsu_10_02_06_38_04.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDcex colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/12 transformation sfGFP-CBDclos colony2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/12 transformation J23114 colony1</td><td>VF 0.75 VR 0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation J23114 colony2</td><td>VF 0.75 VR 0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation J23108 colony1</td><td>VF 0.75 VR 0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 transformation J23108 colony2</td><td>VF 0.75 VR 0.75</td><td>11</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature </td><td>95℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature </td><td>98℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing </td><td>65℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension </td><td>72℃</td><td>30sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>10.12</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>10.12</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>5</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>7</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>8</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>9</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>10</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>11</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>12</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>13</td><td>other sample</td><td>-</td></tr> | ||
+ | <tr><td>14</td><td>2/12 transformation J23114 colony1 /10</td><td></td></tr> | ||
+ | <tr><td>15</td><td>2/12 transformation J23114 colony2 /10</td><td></td></tr> | ||
+ | <tr><td>16</td><td>2/12 transformation J23108 colony1 /10</td><td></td></tr> | ||
+ | <tr><td>17</td><td>2/12 transformation J23108 colony2 /10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6d/T--Kyoto--2_15_No112_Electrophoresis_Notsu_10_01_08_13_13.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1ka ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw</td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--2_15_No114_Gel_Extraction_Notsu_10_01_08_21_26.1.JPG"><img src="https://static.igem.org/mediawiki/2016/3/3b/T--Kyoto--2_15_No114_Gel_Extraction_Notsu_10_01_08_21_26.2.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0215" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/12 transformation J23114 colony2</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>2/12 transformation J23108 colony2</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/15 liquid culture sfGFP-CBDcex colony2</td><td>43.5</td><td>1.91</td><td>1.60</td></tr> | ||
+ | <tr><td>2/15 liquid culture sfGFP-CBDclos colony2</td><td>74.0</td><td>1.86</td><td>1.82</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/16 miniprep sfGFP-CBDcex colony2 /0.3</td><td>cex.Fw /0.75</td><td>VR /0.75</td><td>-</td><td>10.7</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/16 miniprep sfGFP-CBDclos colony2 /0.3</td><td>cex.Fw /0.75</td><td>VR /0.75</td><td>-</td><td>10.7</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw </td><td>48.6</td><td>1.83</td><td>0.19</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>10.7</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>10.7</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/15 liquid culture J23114</td><td>178.8</td><td>1.91</td><td>2.20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/16 PCR m.scFv</td><td>152.6</td><td>1.86</td><td>2.10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Matsumoto, Notsu, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/16 PCR m.scFv</td><td>4</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>11</td><td>30</td></tr> | ||
+ | <tr><td>K592009</td><td>25</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>m.scFv 2/16 EPcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>K592009 2/16EPcut</td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/cc/T--Kyoto--2_16_No122_Gel_Extraction_Notsu_10_01_08_54_18.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0216" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/12 transformation J23108 colony3</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 miniprep BAN-His14 /0.1</td><td>BAN-His-cex.Rv /0.75</td><td>VF /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep BAN-His14 /0.1</td><td>BAN-His-clos.Rv /0.75</td><td>VF /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His12 /0.1</td><td>INP-His-cex.Rv /0.75</td><td>VF /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His12 /0.1</td><td>INP-His-clos.Rv /0.75</td><td>VF /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>25</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Wan, Yamamoto, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/17PCR BAN-His-CBDcexRv+VF</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>2/17PCR BAN-His-CBDclosRv+VF</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>2/17PCR INP-His-CBDcexRv+VF</td><td>25</td></tr> | ||
+ | <tr><td>5</td><td>2/17PCR INP-His-CBDclosRv+VF</td><td>25</td></tr> | ||
+ | <tr><td>6</td><td>2/16PCR sf-GFP-CBDcex</td><td>50</td></tr> | ||
+ | <tr><td>7</td><td>2/16PCR sf-GFP-CBDclos</td><td>50</td></tr> | ||
+ | <tr><td>8</td><td>blank</td><td>-</td></tr> | ||
+ | <tr><td>9</td><td>1kb ladder</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/2c/T--Kyoto--2_17_No8_Gel_Extraction_Notsu_10_02_07_09_31.2..jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Wan, Yamamoto, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/17PCR BAN-His-CBDcexRv+VF</td><td>10.0</td><td>1.86</td><td>0.43</td></tr> | ||
+ | <tr><td>2/17PCR BAN-His-CBDclosRv+VF</td><td>34.6</td><td>1.91</td><td>1.81</td></tr> | ||
+ | <tr><td>2/17PCR INP-His-CBDcexRv+VF</td><td>100.2</td><td>1.90</td><td>1.24</td></tr> | ||
+ | <tr><td>2/17PCR INP-His-CBDclosRv+VF</td><td>116.5</td><td>1.90</td><td>2.08</td></tr> | ||
+ | <tr><td>2/16PCR sf-GFP-CBDcex</td><td>244.0</td><td>1.91</td><td>2.08</td></tr> | ||
+ | <tr><td>2/16PCR sf-GFP-CBDclos</td><td>208.9</td><td>1.90</td><td>2.01</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/17 Gel Extraction BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex /0.5</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>2</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 Gel Extraction BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos /2</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>2</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 Gel Extraction INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex /1</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>3.5</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 Gel Extraction INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos /2</td><td>VF /0.75</td><td>VR /0.75</td><td>-</td><td>0</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Matsumoto, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/16 liquid culture J23108 colony3</td><td>20.8</td><td>1.90</td><td>1.28</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Notsu, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/16 Gel Extraction m.scFv EPcut </td><td>14.3</td><td>1.87</td><td>1.09</td></tr> | ||
+ | <tr><td>2/16 Gel Extraction K592009 EPcut</td><td>14.1</td><td>1.86</td><td>1.60</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/16 Gel Extraction K592009 EPcut /2.5</td><td>2/16 Gel Extraction m.scFv EPcut /5.0</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0217" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/17 ligation m.scFv EPcut+K592009 EPcut</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex</td><td>72.7</td><td>1.87</td><td>1.93</td></tr> | ||
+ | <tr><td>2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos</td><td>112.1</td><td>1.92</td><td>1.34</td></tr> | ||
+ | <tr><td>2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex</td><td>102.4</td><td>1.89</td><td>1.96</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/ac/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_28_40.1.jpg"><img src="https://static.igem.org/mediawiki/2016/7/7e/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_28_40.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos</td><td>103.4</td><td>1.94</td><td>2.04</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/97/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_32_29.1.jpg"><img src="https://static.igem.org/mediawiki/2016/3/30/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_32_29.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>8</td></tr> | ||
+ | <tr><td>2</td><td>2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>8</td></tr> | ||
+ | <tr><td>2</td><td>2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>blank</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>blank</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>BAN-CBDcex</td><td>72.3</td><td>1.85</td><td>1.87</td></tr> | ||
+ | <tr><td>BAN-CBDclos</td><td>79.8</td><td>1.86</td><td>1.26</td></tr> | ||
+ | <tr><td>INP-CBDcex</td><td>83.6</td><td>1.80</td><td>1.57</td></tr> | ||
+ | <tr><td>INP-CBDclos</td><td>39.4</td><td>1.76</td><td>1.77</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>BAN-CBDcex /0.2</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.8</td><td>KAPA Pre Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>BAN-CBDclos /0.2</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.8</td><td>KAPA Pre Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-CBDcex /0.2</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.8</td><td>KAPA Pre Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-CBDclos /0.2</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.8</td><td>KAPA Pre Mix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/17 transformation m.scFv EPcut+K592009 EPcut colony1</td><td>VF 1.25 VR 1.25</td><td>9.5</td><td>2×KAPA HiFi Fast Hotstart /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 transformation m.scFv EPcut+K592009 EPcut colony2</td><td>VF 1.25 VR 1.25</td><td>9.5</td><td>2×KAPA HiFi Fast Hotstart /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 transformation m.scFv EPcut+K592009 EPcut colony3</td><td>VF 1.25 VR 1.25</td><td>9.5</td><td>2×KAPA HiFi Fast Hotstart /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/17 transformation m.scFv EPcut+K592009 EPcut colony4</td><td>VF 1.25 VR 1.25</td><td>9.5</td><td>2×KAPA HiFi Fast Hotstart /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>10sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>55℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0218" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/18 PCR m.scFv EPcut+K592009 EPcut colony1</td><td>49.8</td><td>1.92</td><td>0.08</td></tr> | ||
+ | <tr><td>2/18 PCR m.scFv EPcut+K592009 EPcut colony2</td><td>41.1</td><td>1.81</td><td>1.90</td></tr> | ||
+ | <tr><td>2/18 PCR m.scFv EPcut+K592009 EPcut colony3</td><td>47.2</td><td>1.87</td><td>1.99</td></tr> | ||
+ | <tr><td>2/18 PCR m.scFv EPcut+K592009 EPcut colony4</td><td>47.5</td><td>1.87</td><td>1.93</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>BAN-cex/(25)</td><td>161.1</td><td>1.88</td><td>2.13</td></tr> | ||
+ | <tr><td>BAN-clos/(25)</td><td>217.9</td><td>1.86</td><td>2.10</td></tr> | ||
+ | <tr><td>INP-cex/(25)</td><td>158.6</td><td>1.86</td><td>1.98</td></tr> | ||
+ | <tr><td>INP-clos/(25)</td><td>163.6</td><td>1.85</td><td>1.70</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/19 purification BAN-cex</td><td>12.4</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H/(3)</td><td>0</td><td>12.6</td><td>30</td></tr> | ||
+ | <tr><td>2/19 purification BAN-clos</td><td>9.2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H/(3)</td><td>0</td><td>15.8</td><td>30</td></tr> | ||
+ | <tr><td>2/19 purification INP-cex</td><td>12.6</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H/(3)</td><td>0</td><td>12.4</td><td>30</td></tr> | ||
+ | <tr><td>2/19 purification INP-clos</td><td>12.2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H/(3)</td><td>0</td><td>12.8</td><td>30</td></tr> | ||
+ | <tr><td>2/9 miniprep E1010</td><td>25</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H/(3)</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219” class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Egashira, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>2/19 Reatriction Enzyme Digestion BAN-cex E, Pcut</td><td>30</td></tr> | ||
+ | <tr><td>2</td><td>2/19 Reatriction Enzyme Digestion BAN-clos E, Pcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>2/19 Reatriction Enzyme Digestion INP-cex E, Pcut</td><td>30</td></tr> | ||
+ | <tr><td>0</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/65/T--Kyoto--2_19_No18_Gel_Extraction_Kosaka_09_24_12_03_17.1.jpg"><img src="https://static.igem.org/mediawiki/2016/8/80/T--Kyoto--2_19_No18_Gel_Extraction_Kosaka_09_24_12_03_17.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>2/19 Restriction Enzyme INP-clos EPcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>2/19 Restriction Enzyme E1010 EPcut</td><td>30</td></tr> | ||
+ | <tr><td>4</td><td>blank</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6d/T--Kyoto--2_19_No18_Gel_Extraction_Notsu_10_15_08_27_59.1.jpg"><img src="https://static.igem.org/mediawiki/2016/b/b6/T--Kyoto--2_19_No18_Gel_Extraction_Notsu_10_15_08_27_59.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/19 Restriction Enzyme BAN-cex E, Pcut</td><td>9.6</td><td>1.85</td><td>0.84</td></tr> | ||
+ | <tr><td>〃BAN-clos E, Pcut</td><td>18.9</td><td>1.83</td><td>0.71</td></tr> | ||
+ | <tr><td>〃INP-cex E, Pcut</td><td>14.9</td><td>1.77</td><td>1.46</td></tr> | ||
+ | <tr><td>〃INP-clos E, Pcut</td><td>13.4</td><td>1.73</td><td>1.67</td></tr> | ||
+ | <tr><td>〃E1010 E, Pcut</td><td>19.6</td><td>1.86</td><td>0.40</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan, Egashira, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>insertDNA (µl)</th><th>VectoeDNA (µl)</th><th>ligation high (µl)</th><th>total (µl)</th></tr> | ||
+ | <tr><td>BAN-cex/4</td><td>E1010(pSB1C3)/2</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>BAN-clos/3</td><td>〃/3</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>INP-cex/4.5</td><td>〃/1.5</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>INP-clos/4.5</td><td>〃/1.5</td><td>6</td><td>12</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/19 Ligation BAN-cex+E1010</td><td>10</td><td>DH5a/(20)</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃BAN-clos+E1010</td><td>10</td><td>DH5a/(20)</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃INP-cex+E1010</td><td>10</td><td>DH5a/(20)</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃INP-clos+E1010</td><td>10</td><td>DH5a/(20)</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/18 purification m.scFv EPcut+K592009 EPcut colony1</td><td>12.5</td><td>0.5</td><td>0</td><td>0</td><td>0.5</td><td>10×H /1.5</td><td>0</td><td>0</td><td>15</td></tr> | ||
+ | <tr><td>2/18 purification m.scFv EPcut+K592009 EPcut colony2</td><td>12.5</td><td>0.5</td><td>0</td><td>0</td><td>0.5</td><td>10×H /1.5</td><td>0</td><td>0</td><td>15</td></tr> | ||
+ | <tr><td>2/18 purification m.scFv EPcut+K592009 EPcut colony3</td><td>12.5</td><td>0.5</td><td>0</td><td>0</td><td>0.5</td><td>10×H /1.5</td><td>0</td><td>0</td><td>15</td></tr> | ||
+ | <tr><td>2/18 purification m.scFv EPcut+K592009 EPcut colony4</td><td>12.5</td><td>0.5</td><td>0</td><td>0</td><td>0.5</td><td>10×H /1.5</td><td>0</td><td>0</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1 </td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>2/19 restricion enzyme digestion colony1 /10</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2/19 restricion enzyme digestion colony2 /10</td><td></td></tr> | ||
+ | <tr><td>4</td><td>2/19 restricion enzyme digestion colony3 /10</td><td></td></tr> | ||
+ | <tr><td>5</td><td>2/19 restricion enzyme digestion colony4 /10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/24/T--Kyoto--2_19_No130_Electrophoresis_Notsu_10_01_09_25_34.JPG"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0219" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony4</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0222" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/19 transformation BAN-His-CBDcex colony1</td><td>vf 1.25 vr 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃 colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃 colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>2/19 transformation BAN-His-CBDclos colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃 colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃 colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>2/19 transformation INP-His-CBDcex colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃 colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃 colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0222" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>ladder 1kb/(5)</td><td>/</td></tr> | ||
+ | <tr><td>2</td><td>2/22 PCR BAN-His-CBDcex colony1/(10)</td><td>515</td></tr> | ||
+ | <tr><td>3</td><td>〃colony2/(10)</td><td>515</td></tr> | ||
+ | <tr><td>4</td><td>〃colony3/(10)</td><td>515</td></tr> | ||
+ | <tr><td>5</td><td>2/22 PCR BAN-His-CBDclos colony3/(10)</td><td>487</td></tr> | ||
+ | <tr><td>6</td><td>2/22 PCR INP-His-CBDcex colony1/(10)</td><td>405</td></tr> | ||
+ | <tr><td>7</td><td>〃colony2/(10)</td><td>405</td></tr> | ||
+ | <tr><td>8</td><td>〃colony3/(10)</td><td>405</td></tr> | ||
+ | <tr><td>9</td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e5/T--Kyoto--2_22_No23_Electrophoresis_Kosaka_09_24_12_30_17.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0222" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/22 PCR BAN-His-cex colony1</td><td>CP/5ml</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td></tr> | ||
+ | <tr><td>2/22 PCR BAN-His-clos colony3</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0222" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/19 liquid culture m.scFv EPcut+K592009 EPcut colony2</td><td>137.4</td><td>1.89</td><td>2.12</td></tr> | ||
+ | <tr><td>2/19 liquid culture m.scFv EPcut+K592009 EPcut colony3</td><td>164.0</td><td>1.89</td><td>2.09</td></tr> | ||
+ | <tr><td>2/19 liquid culture m.scFv EPcut+K592009 EPcut colony4</td><td>138.1</td><td>1.86</td><td>2.16</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0223" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>insertDNA (µl)</th><th>VectoeDNA (µl)</th><th>ligation high (µl)</th><th>total (µl)</th></tr> | ||
+ | <tr><td>BAN-cex/4</td><td>E1010cpsb1c3/2</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>BAN-clos/3</td><td>〃/3</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>INP-cex/4.5</td><td>〃/1.5</td><td>6</td><td>12</td></tr> | ||
+ | <tr><td>INP-clos/4.5</td><td>〃/1.5</td><td>6</td><td>12</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0223" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/23 Ligation BAN-cex+E1010</td><td>2</td><td>DH5a/(20)</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃clos〃</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INP-cex〃</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INP-clos〃</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0224" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-CBDclos colony1</td><td>vf1.25 vr1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-CBDcex colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-CBDclos colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0224" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/22 liqid culture BAN-Hi-cex colony1</td><td>45.3</td><td>1.88</td><td>2.08</td></tr> | ||
+ | <tr><td>〃colony2</td><td>47.1</td><td>1.89</td><td>1.98</td></tr> | ||
+ | <tr><td>〃colony3</td><td>43.8</td><td>1.96</td><td>2.08</td></tr> | ||
+ | <tr><td>2/22 BAN-His-clos colony3</td><td>53.7</td><td>1.82</td><td>1.62</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0224" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/23 transformation BAN-clos colony1</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td></tr> | ||
+ | <tr><td>2/23 transformation INP-cex colony2</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td></tr> | ||
+ | <tr><td>2/23 transformation INP-clos colony1</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0225" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-CBDclos colony1</td><td>79.9</td><td>1.89</td><td>2.15</td></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-CBDclos colony2</td><td>68.0</td><td>1.88</td><td>2.02</td></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-CBDclos colony3</td><td>80.7</td><td>1.94</td><td>2.18</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-CBDcex colony2</td><td>106.0</td><td>1.91</td><td>2.17</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-CBDcex colony3</td><td>136.7</td><td>1.93</td><td>2.23</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-CBDclos colony1</td><td>183.7</td><td>1.91</td><td>2.21</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0225" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Sequence</h4></span> | ||
+ | <span class="keln_researcher">Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA template/(µl)</th><th>Primer/(µl)</th><th>MilliQ(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>Bcx 1-F</td><td>2/24 miniprep BAN-His-CBDcex colony1 /6.6</td><td>VF /0.2</td><td>3.2</td><td>10</td></tr> | ||
+ | <tr><td>Bcx 1-R</td><td>2/24 miniprep BAN-His-CBDcex colony1 /6.6</td><td>VR /0.2</td><td>3.2</td><td>10</td></tr> | ||
+ | <tr><td>Bcx 2-F</td><td>2/24 miniprep BAN-His-CBDcex colony2 /6.4</td><td>VF /0.2</td><td>3.4</td><td>10</td></tr> | ||
+ | <tr><td>Bcx 2-R</td><td>2/24 miniprep BAN-His-CBDcex colony2 /6.4</td><td>VR /0.2</td><td>3.4</td><td>10</td></tr> | ||
+ | <tr><td>Bcl1-F</td><td>2/25 miniprep BAN-His-CBDclos colony1 /3.8</td><td>VF /0.2</td><td>6.0</td><td>10</td></tr> | ||
+ | <tr><td>Bcl1-R</td><td>2/25 miniprep BAN-His-CBDclos colony1 /3.8</td><td>VR /0.2</td><td>6.0</td><td>10</td></tr> | ||
+ | <tr><td>Bcl2-F</td><td>2/25 miniprep BAN-His-CBDclos colony3 /3.7</td><td>VF /0.2</td><td>6.1</td><td>10</td></tr> | ||
+ | <tr><td>Bcl2-R</td><td>2/25 miniprep BAN-His-CBDclos colony3 /3.7</td><td>VR /0.2</td><td>6.1</td><td>10</td></tr> | ||
+ | <tr><td>Icx1-F</td><td>2/25 miniprep INP-His-CBDcex colony2 /2.8</td><td>VF /0.2</td><td>7.0</td><td>10</td></tr> | ||
+ | <tr><td>Icx1-R</td><td>2/25 miniprep INP-His-CBDcex colony2 /2.8</td><td>VR /0.2</td><td>7.0</td><td>10</td></tr> | ||
+ | <tr><td>Icx2-F</td><td>2/25 miniprep INP-His-CBDcex colony3 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>Icx2-R</td><td>2/25 miniprep INP-His-CBDcex colony3 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>Icl1-F</td><td>2/25 miniprep INP-His-CBDclos colony1 /1.6</td><td>VF /0.2</td><td>8.2</td><td>10</td></tr> | ||
+ | <tr><td>Icl1-R</td><td>2/25 miniprep INP-His-CBDclos colony1 /1.6</td><td>VR /0.2</td><td>8.2</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Koide --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0225" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/19 transformation BAN-His-cex colony1</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>2/19 transformation BAN-His-cex colony2</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-clos colony1</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>2/23 transformation BAN-His-clos colony3</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>2/23 transformaation INP-His-cex colony2</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>2/23 transformation INP-His-cex colony3</td><td>LB(CP/5ml)</td></tr> | ||
+ | <tr><td>INP-His-clos colony1</td><td>LB(CP/5ml)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0225" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Sequence</h4></span> | ||
+ | <span class="keln_researcher">Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA template/(µl)</th><th>Primer/(µl)</th><th>MilliQ(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>S2-F</td><td>2/22 miniprep scFv colony2 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>S2-R</td><td>2/22 miniprep scFv colony2 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>S3-F</td><td>2/22 miniprep scFv colony3 /1.8</td><td>VF /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>S3-R</td><td>2/22 miniprep scFv colony3 /1.8</td><td>VR /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>S4-F</td><td>2/22 miniprep scFv colony4 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>S4-R</td><td>2/22 miniprep scFv colony4 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0225" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/17 transformaion m.scFv EPcut+K592009 EPcut colony4</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0226" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>J23100</td><td>VF/ 0.75</td><td>J23100+RBSRv</td><td></td><td>11</td><td>PrimeSTARMax Premix/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23108</td><td>VF/ 0.75</td><td>J23108+RBSRv</td><td></td><td>11</td><td>PrimeSTARMax Premix/ 12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23114</td><td>VF/ 0.75</td><td>J23114+RBSRv</td><td></td><td>11</td><td>PrimeSTARMax Premix/ 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0226" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>J23100 iGEM Part</td><td>VF /0.75</td><td>J23100+RBS Rv /0.75</td><td>-</td><td>11</td><td>PrimeSTAR Max Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23108 iGEM Part</td><td>VF /0.75</td><td>J23108+RBS Rv /0.75</td><td>-</td><td>11</td><td>PrimeSTAR Max Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23114 iGEM Part</td><td>VF /0.75</td><td>J23114+RBS Rv /0.75</td><td>-</td><td>11</td><td>PrimeSTAR Max Premix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0226" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>2/26 PCR J23100 /5</td><td>193+α</td></tr> | ||
+ | <tr><td>3</td><td>2/26 PCR J23108 /5</td><td>193+α</td></tr> | ||
+ | <tr><td>4</td><td>2/26 PCR J23114 /5</td><td>193+α</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0226" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>J23100 iGEM Part /0.1</td><td>VF 0.75</td><td>J23100+RBS Rv /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23108 iGEM Part /0.1</td><td>VF 0.75</td><td>J23108+RBS Rv /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | <tr><td>J23114 iGEM Part /0.1</td><td>VF 0.75</td><td>J23114+RBS Rv /0.75</td><td>-</td><td>10.9</td><td>KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0226" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>2/26 PCR J23100</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>2/26 PCR J23108</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>2/26 PCR J23114</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0229" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/26 PCR J23100</td><td>1.5</td><td>2.71</td><td>0.09</td></tr> | ||
+ | <tr><td>2/26 PCR J23108</td><td>3.6</td><td>18.64</td><td>0.01</td></tr> | ||
+ | <tr><td>2/26 PCR J23114</td><td>1.7</td><td>3.05</td><td>0.13</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0229" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>2/29</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Wan, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>J23100 iGEM Part /0.2</td><td>VF /1.5</td><td>J23100+RBS Rv /1.5</td><td>-</td><td>21.8</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | <tr><td>J23108 iGEM Part /0.2</td><td>VF /1.5</td><td>J23108+RBS Rv /1.5</td><td>-</td><td>21.8</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | <tr><td>J23114 iGEM Part /0.2</td><td>VF /1.5</td><td>J23114+RBS Rv /1.5</td><td>-</td><td>21.8</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Li, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>100bp ladder /6</td><td></td></tr> | ||
+ | <tr><td>2,3</td><td>J23114 /50 </td><td>193+α</td></tr> | ||
+ | <tr><td>4,5</td><td>J23100 /50</td><td>193+α</td></tr> | ||
+ | <tr><td>6,7</td><td>J23108 /50</td><td>193+α</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/29 PCR J23114</td><td>4.7</td><td>1.59</td><td>0.01</td></tr> | ||
+ | <tr><td>2/29 PCR J23100</td><td>2.4</td><td>2.60</td><td>0.01</td></tr> | ||
+ | <tr><td>2/29 PCR J23108</td><td>3.8</td><td>4.51</td><td>0.01</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/1 PCR purification J23100 /5</td><td>VF /0.75</td><td>J23100Rv /0.75</td><td>-</td><td>6</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/1 PCR purification J23108 /5</td><td>VF /0.75</td><td>J23108Rv /0.75</td><td>-</td><td>6</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/1 PCR purification J23114 /5</td><td>VF /0.75</td><td>J23114Rv /0.75</td><td>-</td><td>6</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/22 miniprep m.scFv colony3 /0.5</td><td>BAN+scFv /0.75</td><td>VR /0.75</td><td>-</td><td>10.5</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>3/1 PCR J23100 /5</td><td>about 200</td></tr> | ||
+ | <tr><td>3</td><td>3/1 PCR J23108 /5</td><td>about 200</td></tr> | ||
+ | <tr><td>4</td><td>3/1 PCR J23114 /5</td><td>about 200</td></tr> | ||
+ | <tr><td>5</td><td>3/1 PCR m.scFv colony3 /5</td><td>949</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>100bp ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>3/1 OE-PCR J23100 + m.scFv colony3 /2</td><td>about 1200</td></tr> | ||
+ | <tr><td>3</td><td>3/1 OE-PCR J23108 + m.scFv colony3 /2</td><td>about 1200</td></tr> | ||
+ | <tr><td>4</td><td>3/1 OE-PCR J23114 + m.scFv colony3 /2</td><td>about 1200</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/1 OE-PCR J23100 + m.scFv colony3 </td><td>180.5</td><td>1.89</td><td>1.83</td></tr> | ||
+ | <tr><td>3/1 OE-PCR J23108 + m.scFv colony3</td><td>183.3</td><td>1.87</td><td>1.96</td></tr> | ||
+ | <tr><td>3/1 OE-PCR J23114 + m.scFv colony3</td><td>178.8</td><td>1.87</td><td>2.08</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/1 PCR J23100 /1 + 3/1 PCR m.scFv colony3 /0.5</td><td>VF /0.5</td><td>VR /0.75</td><td>-</td><td>9.5</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/1 PCR J23108 /1 + 3/1 PCR m.scFv colony3 /0.5</td><td>VF /0.5</td><td>VR /0.75</td><td>-</td><td>9.5</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/1 PCR J23114 /2 + 3/1 PCR J23114 /0.5</td><td>VF /0.5</td><td>VR /0.75</td><td>-</td><td>8.5</td><td>Prime Star Premix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/1 EPcut J23114 + m.scFv</td><td>20</td></tr> | ||
+ | <tr><td>3</td><td>3/1 EPcut J23108 + m.scFv</td><td>20</td></tr> | ||
+ | <tr><td>4</td><td>3/1 EPcut J23100 + m.scFv</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0301" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Li, Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/1 OE-PCR J23100 + m.scFv colony3</td><td>11</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>-</td><td>14</td><td>30</td></tr> | ||
+ | <tr><td>3/1 OE-PCR J23108 + m.scFv colony3</td><td>11</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>-</td><td>14</td><td>30</td></tr> | ||
+ | <tr><td>3/1 OE-PCR J23114 + m.scFv colony3</td><td>11</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>-</td><td>14</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0302" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/2</h3></span> | ||
+ | <span class="keln_exp"><h4>PartsAwaking</h4></span> | ||
+ | <span class="keln_researcher">Michimori, Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Description</th><th>PartName</th><th>Backbone</th><th>Well</th><th>PlateYear</th><th>PlateNumber</th><th>MilliQ(µl)</th></tr> | ||
+ | <tr><td>f1 origin</td><td>K314110</td><td>pSB1C3</td><td>1A</td><td>2016</td><td>1</td><td>10</td></tr> | ||
+ | <tr><td>T1 terminator from E. coli rrnB</td><td>K731722</td><td>pSB1C3</td><td>1B</td><td>2016</td><td>1</td><td>10</td></tr> | ||
+ | <tr><td>Promoter of the CaiF protein</td><td>K398326</td><td>pSB1C3</td><td>1C</td><td>2016</td><td>1</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0302" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>3/2 Parts Awakening K314110</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/2 Parts Awakening K731722</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/2 Parts Awakening K398326</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0302" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>J23100+m.scFv 3/1 EPcut </td><td>17.7</td><td>1.90</td><td>1.68</td></tr> | ||
+ | <tr><td>J23108+m.scFv 3/1 EPcut </td><td>11.9</td><td>2.04</td><td>0.26</td></tr> | ||
+ | <tr><td>J23114+m.scFv 3/1 EPcut </td><td>16.8</td><td>1.97</td><td>1.79</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0302" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>E0020</td><td>40</td><td>2</td><td>0</td><td>0</td><td>2</td><td>10×H /6</td><td>-</td><td>10</td><td>60</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0302" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>J23100+pSB1C3</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23108+pSB1C3</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23114+pSB1C3</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/9 miniprep E1010 /1</td><td>VF 0.75</td><td>VR 0.75</td><td>-</td><td>10</td><td>Prime Star premix 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/24 miniprep BAN-His-CBDcex colony2 /1</td><td>VF 0.75</td><td>VR 0.75</td><td>-</td><td>10</td><td>Prime Star premix 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 miniprep BAN-His-CBDclos /1</td><td>VF 0.75</td><td>VR 0.75</td><td>-</td><td>10</td><td>Prime Star premix 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDcex /1</td><td>VF 0.75</td><td>VR 0.75</td><td>-</td><td>10</td><td>Prime Star premix 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDclos /1</td><td>VF 0.75</td><td>VR 0.75</td><td>-</td><td>10</td><td>Prime Star premix 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/2 EPcut E0020</td><td>23.1</td><td>1.85</td><td>1.69</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/2 EPcut E0020 (pSB1C3)</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/3 Gel Extraction E0020 EPcut /4</td><td>3/2 Gel Extraction J23100+m.scFv EPcut /12</td><td>16</td><td>32</td></tr> | ||
+ | <tr><td>3/3 Gel Extraction E0020 EPcut /4</td><td>3/2 Gel Extraction J23108+m.scFv EPcut /12</td><td>20</td><td>40</td></tr> | ||
+ | <tr><td>3/3 Gel Extraction E0020 EPcut /4</td><td>3/2 Gel Extraction J23114+m.scFv EPcut /12</td><td>16</td><td>32</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>J23100+PSB1C3</td><td>2</td><td>DH5alpha/ 20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23108+PSB1C3</td><td>2</td><td>DH5alpha/ 20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23114+PSB1C3</td><td>2</td><td>DH5alpha</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0303" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Wan, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>K314110 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>K731722 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>K398326 colony1</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0304" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Li, Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>J23100+pSB1C3</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23108+pSB1C3</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>J23114+pSB1C3</td><td>10</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>2/25 liquid culture BAN-His-CBDcex colony2</td><td>71.4</td><td>1.83</td><td>1.43</td></tr> | ||
+ | <tr><td>2/25 liquid culture BAN-His-CBDclos colony1</td><td>61.6</td><td>1.83</td><td>1.90</td></tr> | ||
+ | <tr><td>2/25 liquid culture INP-His-CBDcex colony3</td><td>83.4</td><td>1.94</td><td>1.92</td></tr> | ||
+ | <tr><td>2/25 liquid culture INP-His-CBDclos colony1</td><td>100.7</td><td>1.87</td><td>1.44</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Wan</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>3/7 miniprep BAN-His-CBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/7 miniprep BAN-His-CBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/7 miniprep INP-His-CBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/7 miniprep INP-His-CBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>3/3 ligation J23100+pSB1C3</td><td></td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/3 ligation J23108+pSB1C3</td><td></td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>3/3 ligation J23114+pSB1C3</td><td></td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/4 transformtion J23100 colony1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/4 transformtion J23100 colony2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/4 transformtion J23108 colony1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/4 transformtion J23108 colony2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/4 transformtion J23114 colony1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/4 transformtion J23114 colony2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>KAPA2G /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>55℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>5sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>10min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Wan, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/7 colony PCR J23100 colony1 /10</td><td>1000+α</td></tr> | ||
+ | <tr><td>3</td><td>3/7 colony PCR J23100 colony2 /10</td><td>1000+α</td></tr> | ||
+ | <tr><td>4</td><td>3/7 colony PCR J23108 colony1 /10</td><td>1000+α</td></tr> | ||
+ | <tr><td>5</td><td>3/7 colony PCR J23108 colony2 /10</td><td>1000+α</td></tr> | ||
+ | <tr><td>6</td><td>3/7 colony PCR J23114 colony1 /10</td><td>1000+α</td></tr> | ||
+ | <tr><td>7</td><td>3/7 colony PCR J23114 colony2 /10</td><td>1000+α</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c9/T--Kyoto--3_7_No154_Electrophoresis_Notsu_10_02_10_08_42.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>5min</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0307" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/7</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/1 purification J23100</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | <tr><td>3/1 purification J23100</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | <tr><td>3/1 purification J23108</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | <tr><td>3/1 purification J23108</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | <tr><td>3/1 purification J23114</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | <tr><td>3/1 purification J23114</td><td>VF /3</td><td>VR /3</td><td>-</td><td>44</td><td>KAPA HiFi /50</td><td>100</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0308" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/7 transformation BAN-CBDcex colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation BAN-CBDcex colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation BAN-CBDclos colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation BAN-CBDclos colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation INP-CBDcex colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation INP-CBDcex colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation INP-CBDclos colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/7 transformation INP-CBDclos colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0308" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/8</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/7 PCR J23100</td><td>423.3</td><td>1.84</td><td>2.20</td></tr> | ||
+ | <tr><td>3/7 PCR J23108</td><td>439.1</td><td>1.85</td><td>2.11</td></tr> | ||
+ | <tr><td>3/7 PCR J23114</td><td>451.2</td><td>1.81</td><td>1.91</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0308" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/8 purification J23100</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23108</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23114</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0308" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Li, Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>J23114 3/8 EPcut</td><td>13.7</td><td>3.62</td><td>0.03</td></tr> | ||
+ | <tr><td>J23108 3/8 EPcut</td><td>15.4</td><td>1.83</td><td>1.14</td></tr> | ||
+ | <tr><td>J23100 3/8 EPcut</td><td>14.5</td><td>1.79</td><td>1.03</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0309" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>K731722</td><td>67.2</td><td>1.70</td><td>1.02</td></tr> | ||
+ | <tr><td>K314110</td><td>28.9</td><td>1.86</td><td>1.14</td></tr> | ||
+ | <tr><td>K398326</td><td>95.6</td><td>1.64</td><td>0.94</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0309" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/1 miniprep K314110</td><td>25</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>3/1 miniprep K731722</td><td>25</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>3/1 miniprep K398326</td><td>25</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0309" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Li</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/8 purification J23100</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23108</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23114</td><td>5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23100</td><td>20</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>5</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23108</td><td>20</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>5</td><td>30</td></tr> | ||
+ | <tr><td>3/8 purification J23114</td><td>20</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>5</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0311" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/11</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>3/10 E.P. cut J23100+scFv</td><td>15</td></tr> | ||
+ | <tr><td>3</td><td>3/10 E.P. cut J23100+scFv</td><td>15</td></tr> | ||
+ | <tr><td>4</td><td>3/10 E.P.cut J23108+scFv</td><td>15</td></tr> | ||
+ | <tr><td>5</td><td>3/10 E.P.cut J23108+scFv</td><td>15</td></tr> | ||
+ | <tr><td>6</td><td>3/10 E.P.cut J23114+scFv</td><td>15</td></tr> | ||
+ | <tr><td>7</td><td>3/10 E.P.cut J23114+scFv</td><td>15</td></tr> | ||
+ | <tr><td>8</td><td>3/9 E.P. cut J23100+scFv</td><td>15</td></tr> | ||
+ | <tr><td>9</td><td>3/9 E.P. cut J23100+scFv</td><td>15</td></tr> | ||
+ | <tr><td>10</td><td>3/9 E.P. cut J23119 scFv</td><td>15</td></tr> | ||
+ | <tr><td>11</td><td>3/9 E.P. cut J23119 scFv</td><td>15</td></tr> | ||
+ | <tr><td>12</td><td>3/9 E.P.cut J23114+scFv</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0311" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/11</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder</td><td>15</td></tr> | ||
+ | <tr><td>2</td><td>3/9 E.P. cut K314110</td><td>15</td></tr> | ||
+ | <tr><td>3</td><td>3/9 E.P. cut K314110</td><td>15</td></tr> | ||
+ | <tr><td>4</td><td>3/9 E.P cut K731722</td><td>15</td></tr> | ||
+ | <tr><td>5</td><td>3/9 E.P cut K731722</td><td>15</td></tr> | ||
+ | <tr><td>6</td><td>3/9 E.P cut K7398326</td><td>15</td></tr> | ||
+ | <tr><td>7</td><td>3/9 E.P cut K7398326</td><td>15</td></tr> | ||
+ | <tr><td>8</td><td>3/9 E.P> cut J23114+scFv</td><td>15</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/9a/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C1.jpg"> <img src="https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C2.jpg"> <img src="https://static.igem.org/mediawiki/2016/8/8c/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C3.jpg"> <img src="https://static.igem.org/mediawiki/2016/c/ce/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C4.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0311" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/11</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Yoshimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/11 Gel Ex J23108+scFv</td><td>18.9</td><td>1.90</td><td>0.19</td></tr> | ||
+ | <tr><td>3/11 Gel Ex J23108+scFv</td><td>7.0</td><td>1.92</td><td>0.57</td></tr> | ||
+ | <tr><td>3/11 Gel Ex J23114+scFv</td><td>5.3</td><td>2.37</td><td>0.25</td></tr> | ||
+ | <tr><td>3/11 Gel Ex J23100+scFv</td><td>39.2</td><td>1.93</td><td>1.29</td></tr> | ||
+ | <tr><td>3/11 Gel Ex J23108+scFv</td><td>33.6</td><td>1.82</td><td>0.68</td></tr> | ||
+ | <tr><td>3/11 Gel Ex J23114+scFv</td><td>27.8</td><td>1.86</td><td>1.25</td></tr> | ||
+ | <tr><td>3/11 Gel Ex pSCIC3</td><td>29.0</td><td>1.86</td><td>1.42</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0311" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/11</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/11 Gel Extraction PSB1C3/ 5</td><td>3/11 Gel Extraction J23100+scfv/ 5.6</td><td>10.6</td><td>21.2</td></tr> | ||
+ | <tr><td>3/11 Gel Extraction PSB1C3/ 5</td><td>3/11 Gel Extraction J23108+scfv/ 5</td><td>11.5</td><td>23</td></tr> | ||
+ | <tr><td>3/11 Gel Extraction PSB1C3/ 5</td><td>3/11 Gel Extraction J23114+scfv/ 5</td><td>12.8</td><td>25.6</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0311" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/11</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>3/11 Ligation PSB1C3+J23100+scFV</td><td></td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>3/11 Ligation PSB1C3+J23108+scFv</td><td></td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | <tr><td>3/11 Ligation PSB1C3+J23114+scFv</td><td></td><td>DH5α /20</td><td>LB</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/8 PCR BANcex colony1</td><td>515</td></tr> | ||
+ | <tr><td>3</td><td>3/8 PCR BAN-cex colony2</td><td>515</td></tr> | ||
+ | <tr><td>4</td><td>3/8 PCR BAN-clos colony1</td><td>487</td></tr> | ||
+ | <tr><td>5</td><td>3/8 PCR BAN-clos-colony2</td><td>487</td></tr> | ||
+ | <tr><td>6</td><td>3/8 PCR INP-cex colony 1</td><td>1405</td></tr> | ||
+ | <tr><td>7</td><td>3/8 PCR INP-cex colony 2</td><td>1405</td></tr> | ||
+ | <tr><td>8</td><td>3/8 PCR INP-clos colony 1</td><td>1378</td></tr> | ||
+ | <tr><td>9</td><td>3/8 PCR INP-clos colony 2</td><td>1378</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Kyoto--3_14_No43_Electrophoresis_Yoshimoto_09_02_04_10_33.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4> PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>VF 0.75 CBDcex ctl. Rv0.75</td><td>11</td><td>KAPA HiFi 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>CBDcex ctl Fw 0.75 VR0.75</td><td>11</td><td>KAPA HiFi 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/18 INPclos Gel Extraction</td><td>VF 0.75 CBD clos ctl. Rv0.75</td><td>11</td><td>KAPA HiFi 12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/18 INPclos Gel Extraction</td><td>CBDclos ctl Fw 0.75 VR0.75</td><td>11</td><td>KAPA HiFi 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>4</td><td>3/14PCR INP cex (VF CBD cex ctl.Rv)25</td><td>5</td></tr> | ||
+ | <tr><td>5</td><td>3/14PCR INPcex (CBD cex ctl.Fw VR) 25</td><td>5</td></tr> | ||
+ | <tr><td>6</td><td>3/14PCR INP clos(VF CBDclos ctl.Rv)25</td><td>5</td></tr> | ||
+ | <tr><td>7</td><td>3/14PCR INPclos (CBDclos ctl.Fw VR) 25</td><td>5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--3_14_No45_Gel_Extraction_Yoshimoto_09_02_09_48_51.1.jpg"><img src="https://static.igem.org/mediawiki/2016/5/56/T--Kyoto--3_14_No45_Gel_Extraction_Yoshimoto_09_02_09_48_51.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/7 miniprep BAN-His-CBDcex colony2</td><td>30</td><td>-</td><td>-</td><td>1</td><td>1</td><td>4</td><td>-</td><td>4</td><td>40</td></tr> | ||
+ | <tr><td>3/7miniprep BAN-His-CBDclos colony1</td><td>30</td><td>-</td><td>-</td><td>1</td><td>1</td><td>4</td><td>-</td><td>4</td><td>40</td></tr> | ||
+ | <tr><td>3/7miniprep INP-His-CBDcex colony3</td><td>30</td><td>-</td><td>-</td><td>1</td><td>1</td><td>4</td><td>-</td><td>4</td><td>40</td></tr> | ||
+ | <tr><td>3/'miniprep INP-His-CBDcloscolony 1</td><td>40</td><td>-</td><td>-</td><td>1</td><td>1</td><td>5</td><td>-</td><td>3</td><td>50</td></tr> | ||
+ | <tr><td>2/9 miniprep J23105</td><td>61</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>70</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>OE-PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/14 Gel Ex INP cex 0.5 VR ct Fw + 3/14 Gel Ex INP cex 0.5 VF ct Rv</td><td>VF1.5 VR1.5</td><td>21</td><td>25</td><td>50</td></tr> | ||
+ | <tr><td>3/14 Gel Ex INP clos 0.5 VR ct Fw + 3/14 Gel Ex INP clos 0.5 VF ct Rv</td><td>VF1.5 VR1.5</td><td>21</td><td>25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>78℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>45sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Uchino --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 INP His9</td><td>VF 0.75 scFv ctl. Rv 0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 INP His9</td><td>scFv ctl Fw 0.75 VR 0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>VF 0.75 CBDcex ctl. Rv0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>CBDcex ctl. Fw 0.75 VR 0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/18 INPclos Gel Extraction</td><td>CBDclos ctl. Rv 0.75 VF 0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/18 INPclos Gel Extraction</td><td>CBDclos ctl Fw 0.75</td><td>11</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0314" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>3/14 PCR INP His9 (VF scFv.ctl. Rv)</td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>3/14 PCR INP His9 (scFv ctl.Fw VR)</td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/11/T--Kyoto--3_14_No173_Gel_Extraction_Yoshimoto_09_16_10_54_56%2C1.jpg"><img src="https://static.igem.org/mediawiki/2016/c/ce/T--Kyoto--3_14_No173_Gel_Extraction_Yoshimoto_09_16_10_54_56%2C2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0315" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/14 OEPCR CBDcex ctl 50</td><td>10</td></tr> | ||
+ | <tr><td>3</td><td>3/14 OEPCR CBDclos ctl 50</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0315" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>VF /1.5</td><td>CBDcex.ctl.RV /1.5</td><td>-</td><td>22</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPcex colony2</td><td>CBDcex.ctl.Fw /1.5</td><td>VR /1.5</td><td>-</td><td>22</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPclos colony1</td><td>VF /1.5</td><td>CBDclos.ctl.Rv /1.5</td><td>-</td><td>22</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPclos colony1</td><td>CBDclos.ctl.Fw /1.5</td><td>VR /1.5</td><td>-</td><td>22</td><td>KAPA HiFi /25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0315" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>4</td><td>3/15 PCR INPcex(VF 20, CBDcex ctl.Rv 20)</td><td>4</td></tr> | ||
+ | <tr><td>5</td><td>3/15 PCR INPcex(CBDcex ctl.Fw20, VR 20)</td><td>4</td></tr> | ||
+ | <tr><td>6</td><td>3/15 PCR INPclos(VF 20 CBDclos ctl.Rv 20)</td><td>4</td></tr> | ||
+ | <tr><td>7</td><td>3/15 PCR INPclos(CBDclos ctl.Fw20, VR 20)</td><td>4</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0315" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Li, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>3/11 scFv J23100 colony1,4 colony2,3(plate2)</td><td>LB(CP) 5ml×2</td></tr> | ||
+ | <tr><td>3/11 scFv J23108 colony 2(plate1) colony(plate2)</td><td>LB(CP) 5ml×2</td></tr> | ||
+ | <tr><td>3/14 scFv J23114 colony 1,2,4(plate1) colony1,2,(plate2)</td><td>LB(CP) 5ml×2</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/15 Gelex CBDcex ctl. Fw VR</td><td>55.3</td><td>1.95</td><td>0.11</td></tr> | ||
+ | <tr><td>3/15 Gelex CBDcex VF ctl.RV</td><td>8.2</td><td>1.74</td><td>1.04</td></tr> | ||
+ | <tr><td>3/15Gelex CBDclos ctl Fw VR</td><td>41.2</td><td>1.85</td><td>1.51</td></tr> | ||
+ | <tr><td>3/15Gelex CBDclos VF ctl RV</td><td>10.6</td><td>1.56</td><td>0.60</td></tr> | ||
+ | <tr><td>3/15Gelex BANcexSP</td><td>34.9</td><td>1.70</td><td>1.84</td></tr> | ||
+ | <tr><td>3/15Gelex BANclosSP</td><td>32.0</td><td>1.95</td><td>0.07</td></tr> | ||
+ | <tr><td>3/15 Gelex INPcex SP</td><td>40.3</td><td>1.76</td><td>1.76</td></tr> | ||
+ | <tr><td>3/15Gelex INPclosSP</td><td>106.8</td><td>1.83</td><td>0.19</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>3</td><td>3/15 PCR INPcex(VF 20, CBDcex ctl, RV 20)</td><td>4</td></tr> | ||
+ | <tr><td>4</td><td>3/15 PCR INPclos(VF 20, CBDclos ctl, RV 20)</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>4</td><td>CBDcex ctl</td><td></td></tr> | ||
+ | <tr><td>5</td><td>CBDclos ctl</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/15 Gel Ex INPcex 0.5(VF ctl.RV) 3/15 Gel Ex INPcex0.2(ctl.Fw VR)</td><td>VF1.25</td><td>VR1.25</td><td>KAPA taq 12.5</td><td>9.3</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/15 GEl EX INPclos 0.5(VF ctl RV) 3/15Gel Ex INPclos0.2(ctl.Fw VR)</td><td>VF1.25</td><td>VR1.25</td><td>KAPA taq 12.5</td><td>9.3</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/14 Gel Extraction INP-His9 VR/ct/Rv /0.5 + 3/14 Gel Extraction INP-His9 /0.5 </td><td>VF /1.5</td><td>VR /1.5</td><td>-</td><td>21</td><td>2×KAPA HiFi /25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>KAPA HiFi/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 INP HIs9</td><td>VF 1.5 scFvctl. RV1.5</td><td>22</td><td>25</td><td>50</td></tr> | ||
+ | <tr><td>2/8 INP His9</td><td>VR1.5 scFvctl. Fw 1.5 VR1.5</td><td>22</td><td>25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>J23100 scFv c1-p1</td><td>135.7</td><td>1.85</td><td>1.76</td></tr> | ||
+ | <tr><td>J23100 scFv C4-p1</td><td>135.1</td><td>1.86</td><td>1.85</td></tr> | ||
+ | <tr><td>J23100 scFv c3-p2</td><td>155.0</td><td>1.87</td><td>2.04</td></tr> | ||
+ | <tr><td>J23100 scFv c3-p2</td><td>205.9</td><td>1.76</td><td>1.32</td></tr> | ||
+ | <tr><td>J23108 scFv c2-p1</td><td>166.5</td><td>1.88</td><td>2.06</td></tr> | ||
+ | <tr><td>J23108 scFvc1-p2</td><td>95.7</td><td>1.84</td><td>1.83</td></tr> | ||
+ | <tr><td>J23114 scFv c1-p1</td><td>145.7</td><td>1.80</td><td>1.99</td></tr> | ||
+ | <tr><td>J23114 scFv c2-p1</td><td>111.7</td><td>1.90</td><td>2.16</td></tr> | ||
+ | <tr><td>J23114 scFv c4-p1</td><td>169.2</td><td>1.90</td><td>2.11</td></tr> | ||
+ | <tr><td>J23114 scFv c1-p2</td><td>127.4</td><td>1.87</td><td>2.00</td></tr> | ||
+ | <tr><td>J23114 scFv c2-p2</td><td>136.2</td><td>1.90</td><td>2.13</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sukegawa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Sequence</h4></span> | ||
+ | <span class="keln_researcher">Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA template/(µl)</th><th>Primer/(µl)</th><th>MilliQ(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>100A-F</td><td>3/15 miniprep J23100 scFv c1-p1 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>100A-R</td><td>3/15 miniprep J23100 scFv c1-p1 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>100B-F</td><td>3/15 miniprep J23100 scFv c4-p1 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>100b-R</td><td>3/15 miniprep J23100 scFv c4-p1 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>100C-F</td><td>3/15 miniprep J23100 scFv c2-p2 /1.9</td><td>VF /0.2</td><td>7.9</td><td>10</td></tr> | ||
+ | <tr><td>100C-R</td><td>3/15 miniprep J23100 scFv c2-p2 /1.9</td><td>VR /0.2</td><td>7.9</td><td>10</td></tr> | ||
+ | <tr><td>100D-F</td><td>3/15 miniprep J23100 scFv c3-p2 /1.5</td><td>VF /0.2</td><td>8.3</td><td>10</td></tr> | ||
+ | <tr><td>100D-R</td><td>3/15 miniprep J23100 scFv c3-p2 /1.5</td><td>VR /0.2</td><td>8.3</td><td>10</td></tr> | ||
+ | <tr><td>108A-F</td><td>3/15 miniprep J23108 scFv c2-p1 /1.8</td><td>VF /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>108A-R</td><td>3/15 miniprep J23108 scFv c2-p1 /1.8</td><td>VR /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>108B-F</td><td>3/15 miniprep J23108 scFv c1-p2 /3.1</td><td>VF /0.2</td><td>6.7</td><td>10</td></tr> | ||
+ | <tr><td>108B-R</td><td>3/15 miniprep J23108 scFv c1-p2 /3.1</td><td>VR /0.2</td><td>6.7</td><td>10</td></tr> | ||
+ | <tr><td>114A-F</td><td>3/15 miniprep J23114 scFv c1-p1 /2.1</td><td>VF /0.2</td><td>7.7</td><td>10</td></tr> | ||
+ | <tr><td>114A-R</td><td>3/15 miniprep J23114 scFv c1-p1 /2.1</td><td>VR /0.2</td><td>7.7</td><td>10</td></tr> | ||
+ | <tr><td>114B-F</td><td>3/15 miniprep J23114 scFv c2-p1 /2.7</td><td>VF /0.2</td><td>7.1</td><td>10</td></tr> | ||
+ | <tr><td>114B-R</td><td>3/15 miniprep J23114 scFv c2-p1 /2.7</td><td>VR /0.2</td><td>7.1</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Sequence</h4></span> | ||
+ | <span class="keln_researcher">Michimori</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA template/(µl)</th><th>Primer/(µl)</th><th>MilliQ(µl)</th><th>Total(µl)</th></tr> | ||
+ | <tr><td>114C-F</td><td>3/15 miniprep J23114 scFv c4-p1 /1.8</td><td>VF /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>114C-R</td><td>3/15 miniprep J23114 scFv c4-p1 /1.8</td><td>VR /0.2</td><td>8.0</td><td>10</td></tr> | ||
+ | <tr><td>114C-F</td><td>3/15 miniprep J23114 scFv c1-p2 /2.4</td><td>VF /0.2</td><td>7.4</td><td>10</td></tr> | ||
+ | <tr><td>114C-R</td><td>3/15 miniprep J23114 scFv c1-p2 /2.4</td><td>VR /0.2</td><td>7.4</td><td>10</td></tr> | ||
+ | <tr><td>114C-F</td><td>3/15 miniprep J23114 scFv c2-p2 /2.2</td><td>VF /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | <tr><td>114C-R</td><td>3/15 miniprep J23114 scFv c2-p2 /2.2</td><td>VR /0.2</td><td>7.6</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sukegawa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>2</td><td>3/15 PCR INP His9(VF scFv CH. Rv)</td><td>20</td></tr> | ||
+ | <tr><td>3</td><td>3/15 PCR INP His9 (scFv ctl, Fw VR)</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>3/15 PCR INP His9 (VF scFv ctl RV)</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sukegawa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>scFv (VF ctl.Rv)</td><td></td></tr> | ||
+ | <tr><td>3</td><td>scFv ctl</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>OEPCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample1/(µl)</th><th>Sample2/(µl)</th><th>Primer/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/15 Gel Extraction scfv (VF ctl.Rv)/ 0.5</td><td>3/15 Gel Extraction scfv (ctl.Fw VR)</td><td>VF/ 1.25, VR/1.25</td><td>-</td><td>9.3</td><td>KAPA Taq/ 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>scFv (VF ctl.RV) 0.5</td><td>VF 1.25 ctl.Rv) 1.25</td><td>9.5</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0316" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/16 Gel Extraction scFv(VF.ctl.Rv) /0.25+3/16 Gel Extraction scFv(ctl.Fw.VR) /0.25</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.5</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/16 Gel Ex scFv(VF ctl.Rv)0.25, 3/15 Gel EX CBDcex(ctl.Fw VR) 0.25</td><td>VF 1.25</td><td>VR1.25</td><td>KAPA Taq 12.5</td><td>9.5</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/16 Gel Ex scFv(VF ctl.Rv) 0.25, Gel Ex CBDclos(ctl,Fw VR) 0.25</td><td>VF 1.25</td><td>VR1.25</td><td>KAPA Taq 12.5</td><td>9.5</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>3</td><td>3/16 OEPCR CBDcex ctl 25</td><td>500</td></tr> | ||
+ | <tr><td>4</td><td>3/16 OEPCR CBDclos ctl 25</td><td>500</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/84/T--Kyoto--3_17_No59_Electrophoresis_Yoshimoto_09_03_12_05_46.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>2</td><td>CBDcex ctl 25 </td><td></td></tr> | ||
+ | <tr><td>3</td><td>CBDclos ctl 25</td><td></td></tr> | ||
+ | <tr><td>4</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/16 Gel Ex scFv(VF ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1</td><td>VF 1.25</td><td>VR1.25</td><td>KAPA 12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/16 Gel Ex scFv(VF ctl,Rv) 0.1, 3/15 GelEx CBDclos(ctl,Fw VR) 0.1 ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1</td><td>VF 1.25</td><td>VR1.25</td><td>KAPA 12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/8 Liquid Culture BAN-His-cex</td><td>39.3</td><td>1.91</td><td>1.67</td></tr> | ||
+ | <tr><td>3/8 Liquid Culture BAN-His=clos</td><td>45.1</td><td>1.84</td><td>1.44</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>95℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>15sec</td><td>40</td></tr> | ||
+ | <tr><td>Annealing</td><td>55℃</td><td>15sec</td><td>40</td></tr> | ||
+ | <tr><td>Elongation</td><td>72℃</td><td>1min</td><td>40</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>1min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/17 miniprep BAN-His-cex</td><td>CBDcexFw 1.25</td><td>VR1.25</td><td>12.5</td><td>9</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/17 miniprep BAN-His-clos</td><td>CBDclosFw 1.25</td><td>VR1.25</td><td>12.5</td><td>9</td><td></td><td>25</td></tr> | ||
+ | <tr><td>2/9 miniprep J23105_1</td><td>VF 1.25</td><td>RFP-cex RV 1.25</td><td>12.5</td><td>9</td><td></td><td>25</td></tr> | ||
+ | <tr><td>2/9 miniprep J23105_1</td><td>VF 1.25</td><td>RFP-clos Rv 1.25</td><td>12.5</td><td>9</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/16 OEPCR scFv ctl/ 25</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0317" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/17</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/16 Gel Extraction scFv(VF.ctl.Rv) /0.1+3/15 Gel Extraction scFv(ctl.Fw.VR) /0.1</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>9.8</td><td>2×KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>3/10 PCR BAN-cex CBDcexFW VR </td><td>25</td></tr> | ||
+ | <tr><td>3</td><td>3/10 PCR BAN-clos CBDclosFw VR </td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>3/10 PCR J23105 VF RFPcexRv </td><td>25</td></tr> | ||
+ | <tr><td>5</td><td>3/10 PCRJ23105 VF RFPclos </td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/62/T--Kyoto--3_18_No63_Gel_Extraction_Yoshimoto_09_03_12_29_48.1.jpg"><img src="https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--3_18_No63_Gel_Extraction_Yoshimoto_09_03_12_29_48.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/17 PCR BAN-CBDcex CBDcex.Fw+Rv</td><td>127.7</td><td>1.90</td><td>1.57</td></tr> | ||
+ | <tr><td>3/17 PCR BAN-CBDclos CBDclos.Fw+VR</td><td>93.4</td><td>1.92</td><td>2.19</td></tr> | ||
+ | <tr><td>3/17 PCR J23105 VF+RFP-CBDcex.Rv</td><td>104.0</td><td>1.84</td><td>1.50</td></tr> | ||
+ | <tr><td>3/17 PCR J23105 VF+RFP-CBDclos.Rv</td><td>123.0</td><td>1.87</td><td>1.84</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>95℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>15sec</td><td>40</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>40</td></tr> | ||
+ | <tr><td>Elongation</td><td>72℃</td><td>1min</td><td>40</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>1min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/18 Gel Extraction BAN-His-cex 0.1 cexFw+VR , 3/18Gel Extraction J23105 VF+RFP-cexRv</td><td>VF 1.25</td><td>VR 1.25</td><td>KAPATaq 12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/18 Gel Extraction BAN-His-clos 0.1 closFw+VR , 3/18Gel Extraction J23105 VF+RFP-closRv</td><td>VF 1.25</td><td>VR 1.25</td><td>KAPATaq 12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3/18 OEPCR RFP-cex</td><td></td></tr> | ||
+ | <tr><td>3</td><td>3/18 OEPCR RFP-clos</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/69/T--Kyoto--3_18_No66_Gel_Extraction_Yoshimoto_09_04_05_26_17.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4> PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/25 INP cex colony2 0.1×2</td><td>VF 0.75×2 ctl RV 0.75</td><td>10.9×2</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP cex colony2 0.1×2</td><td>ctl Fw 0.75×2 VR 0.75×2</td><td>10.9×2</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP cex colony2 0.1×2</td><td>VF 0.75×2 VR 0.75×2</td><td>10.9×2</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP cex colony2 0.1×2</td><td>nothing</td><td>12.4</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP clos colony1 0.1</td><td>VF ctl.Rv 0.75×2</td><td>10.9</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP clos colony1 0.1</td><td>ctl.Rv 0.75×2</td><td>10.9</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP clos colony1 0.1</td><td>VF 0.75×2 VR 0.75×2</td><td>10.9</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | <tr><td>2/25 INP clos colony1 0.1</td><td>nothing</td><td>12.4</td><td>12.5×2</td><td>25×2</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INP-His=CNDcex+RFP colony 1(3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-CBDcex+RFP colony (3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-CBDclos+RFP colony1(3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-CBDclos+RFP colony 2(3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-CBDcex+RFP colony1( 3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BAN-His-CBDcex+RFP colony2 (3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BAN-His-CBDclos+RFP colony1(3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BAN-His-CBDclos+RFP colony2(3/16 ligation product)</td><td>VF 1.25, VR .125</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kim, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>1 kb ladder 5</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2/25 INPcex colony2 10</td><td></td></tr> | ||
+ | <tr><td>4</td><td>2/25 INPcex colony2 10</td><td></td></tr> | ||
+ | <tr><td>5</td><td>2/25 INPcex colony2 10</td><td></td></tr> | ||
+ | <tr><td>6</td><td>2/25 INPcex colony210</td><td></td></tr> | ||
+ | <tr><td>7</td><td>2/25 INPclos colony1 10</td><td></td></tr> | ||
+ | <tr><td>8</td><td>2/25 INPclos colony1 10</td><td></td></tr> | ||
+ | <tr><td>9</td><td>2/25 INPclos colony1 10</td><td></td></tr> | ||
+ | <tr><td>10</td><td>2/25 INPclos colony1 10</td><td></td></tr> | ||
+ | <tr><td>11</td><td>100bp ladder 5</td><td></td></tr> | ||
+ | <tr><td>12</td><td>1kbp ladder 5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/88/T--Kyoto--3_18_No69_Electrophoresis_Yoshimoto_09_04_05_54_18.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0318" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/18</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INP HIs9 0.1x2</td><td>VF 0.75x2 ctl. Rv 0.75x2</td><td>10.9x2</td><td>12.5x2</td><td>25x2</td></tr> | ||
+ | <tr><td>INP HIs9 0.1x2</td><td>ctl.Fw 1.75 VR 0.75</td><td>10.9x2</td><td>12.5x2</td><td>25x2</td></tr> | ||
+ | <tr><td>INP HIs9 0.1x2</td><td>VF 0.75 VR 0.75</td><td>10.9x2</td><td>12.5x2</td><td>25x2</td></tr> | ||
+ | <tr><td>INP HIs9 0.1x2</td><td>nothing</td><td>12.4</td><td>12.5x2</td><td>25x2</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /7</td><td></td></tr> | ||
+ | <tr><td>2</td><td>INP-His-CBDcex+RFP colony1 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>3</td><td>INP-His-CBDcex+RFP colony2 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>4</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>5</td><td>INP-His-CBDclos+RFP colony1 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>6</td><td>INP-His-CBDclos+RFP colony2 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>7</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>8</td><td>BAN-His-CBDcex+RFP colony1 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>9</td><td>BAN-His-CBDcex+RFP colony2 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>10</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>11</td><td>BAN-His-CBDclos+RFP colony1 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>12</td><td>BAN-His-CBDclos+RFP colony2 (3/19 PCR) /20</td><td></td></tr> | ||
+ | <tr><td>13</td><td>blank</td><td></td></tr> | ||
+ | <tr><td>14</td><td>100bp ladder /7</td><td></td></tr> | ||
+ | <tr><td>15</td><td>-</td><td></td></tr> | ||
+ | <tr><td>16</td><td>-</td><td></td></tr> | ||
+ | <tr><td>17</td><td>-</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/70/T--Kyoto--3_22_No72_Electrophoresis_Notsu_10_16_12_57_19.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>7</td><td>2/25 INPcexcolony2 (5) 10</td><td></td></tr> | ||
+ | <tr><td>8</td><td>2/25 INPcexcolony2 (6) 10</td><td></td></tr> | ||
+ | <tr><td>9</td><td>2/25 INPcexcolony2 (7) 10</td><td></td></tr> | ||
+ | <tr><td>10</td><td>2/25 INPcexcolony2 (8) 10</td><td></td></tr> | ||
+ | <tr><td>11</td><td>2/25 INPclos colony1(9) 10</td><td></td></tr> | ||
+ | <tr><td>12</td><td>2/25 INPclos colony1(10) 10</td><td></td></tr> | ||
+ | <tr><td>13</td><td>2/25 INPclos colony1(11) 10</td><td></td></tr> | ||
+ | <tr><td>14</td><td>2/25 INPclos colony1(12) 10</td><td></td></tr> | ||
+ | <tr><td>16</td><td>100b ladder 5</td><td></td></tr> | ||
+ | <tr><td>17</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>5</td><td>3/22 PCR CBDcex(VF/ctl.Rv) 10</td><td></td></tr> | ||
+ | <tr><td>6</td><td>3/22 PCR CBDcex(ctl.Fw/VR) 10</td><td></td></tr> | ||
+ | <tr><td>7</td><td>3/22 PCR CBDclos(VP/ctl.RV) 10</td><td></td></tr> | ||
+ | <tr><td>8</td><td>3/22 PCR CBDclos(ctl.Fw/VR) 10</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/25 INPcex colony2 0.1</td><td>VF 1.5 ctl, Rv 1.5</td><td>21.9</td><td>25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPcex colony2 0.1</td><td>ctl.Fw 1.5 ,VR 1.5</td><td>21.9</td><td>25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPclos colony1 0.1</td><td>VF 1.5, ctl,Rv 1.5</td><td>21.9</td><td>25</td><td>50</td></tr> | ||
+ | <tr><td>2/25 INPclos colony1 0.1</td><td>ctl,Fw 1.5 VR 1.5</td><td>21.9</td><td>25</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Notsu, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>BANcex+RFP col.1</td><td>147.8</td><td>1.89</td><td>2.18</td></tr> | ||
+ | <tr><td>BANcex+RFP col.2 </td><td>133.9</td><td>1.87</td><td>2.18</td></tr> | ||
+ | <tr><td>BANcex+RFP col.1</td><td>132.4</td><td>1.85</td><td>2.19</td></tr> | ||
+ | <tr><td>BANclos+RFP col.2</td><td>126.4</td><td>1.85</td><td>2.22</td></tr> | ||
+ | <tr><td>INPcex+RFP col.1</td><td>179.3</td><td>1.87</td><td>2.18</td></tr> | ||
+ | <tr><td>INPcex+RFP col.2</td><td>144.9</td><td>1.87</td><td>2.19</td></tr> | ||
+ | <tr><td>INPclos+RFPcol.1</td><td>152.2</td><td>1.87</td><td>2.23</td></tr> | ||
+ | <tr><td>INPclos+RFPcol.2</td><td>152.4</td><td>1.86</td><td>2.19</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Koide --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 INP-His9 0.1</td><td>VF 1.5</td><td>ctl.Rv 1.5</td><td>25</td><td>21.9</td><td></td><td>50</td></tr> | ||
+ | <tr><td>2/8 INP-His9 0.1</td><td>scFv ctl.Fw 1.5</td><td>VR 1.5</td><td>25</td><td>21.9</td><td></td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Egashira, Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/22 PCR scFv (VF/ctl.RN)</td><td>105.4</td><td>1.81</td><td>1.54</td></tr> | ||
+ | <tr><td>3/22 PCR scFv (ctl. FV/VR)</td><td>127.4</td><td>1.87</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Iguchi --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0322" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/22</h3></span> | ||
+ | <span class="keln_exp"><h4>OEPCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample1/(µl)</th><th>Sample2/(µl)</th><th>Primer/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/23 Gel Extraction scfv (VF ctl.Rv)/ 0.1</td><td>3/23 Gel Extraction scfv (ctl.Fw VR)/ 0.1</td><td>VF/ 1.25, VR/1.25</td><td>-</td><td>10.8</td><td>KAPA Taq/ 12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>5</td><td>3/22 PCR CBDcex(VF/ctl.Rv) 40</td><td></td></tr> | ||
+ | <tr><td>6</td><td>3/22 PCR CBDcex(ctl.Fw/VR) 40</td><td></td></tr> | ||
+ | <tr><td>7</td><td>3/22 PCR CBDclos(VF/ctl.Rv) 40</td><td></td></tr> | ||
+ | <tr><td>8</td><td>3/22 PCR CBDclos(ctl.Fw/VR) 40</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>95℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>35</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Elongation</td><td>72℃</td><td>15sec</td><td>35</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>10℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/18 Gelex BANcex Fw+VR 0.1, 3?18 Gelex J23105 VF+cexRv 0.1</td><td>VF1.25 </td><td>VR 1.25</td><td>12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/18 Gelex BANclos closFV VR 0.1, 3/18Gelex J23105 VF+closR 0.1</td><td>VF 1.25 </td><td>VR 1.25</td><td>12.5</td><td>9.8</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction (Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/22 PCR CBDcex(ctlFw/VR)</td><td>26.9</td><td>1.75</td><td>1.04</td></tr> | ||
+ | <tr><td>3/22PCR CBDclos(ctl Fw/VR)</td><td>100.6</td><td>1.90</td><td>1.65</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>3</td><td>3/18 OEPCR BANcex cexFw+VR J23105 VF+cexRv 25</td><td></td></tr> | ||
+ | <tr><td>4</td><td>BANclos clos Fw+VR J23105 VF+clos 25</td><td></td></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 10</td><td></td></tr> | ||
+ | <tr><td>2</td><td>100bp ladder 10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Kyoto--3_23_No78_Electrophoresis_Yoshimoto_09_04_06_47_22.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>4</td><td>3/23 OEPCR CBDcex ctl 25</td><td></td></tr> | ||
+ | <tr><td>5</td><td>3/23 OEPCR CBDclos ctl 25</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/23 Gel Ex scFv(VF/ctl.Rv) 0.1, 3/23 Gel Ex CBDcex(ctl.Fw/VR) 0.1</td><td>VF 0.75 VR 0.75</td><td>10.8</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>3/23 Gel Ex scFv (VF/ctl.Rv), 3/23 Gel Ex clos(ctl.Fw/VR)</td><td>VF 0.75 VR 0.75</td><td>10.8</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Koide --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0323" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder 5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>100b ladder 7.5</td><td></td></tr> | ||
+ | <tr><td>3</td><td>3/22 PCR scFv(VP/ctl.Rv) 40</td><td></td></tr> | ||
+ | <tr><td>4</td><td>3/22 PCR scFv(ctl.Fw/VR)</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--3_23_No199_Electrophoresis_Koide_09_19_03_50_56%2C1.jpg"><img src="https://static.igem.org/mediawiki/2016/c/c4/T--Kyoto--3_23_No199_Electrophoresis_Koide_09_19_03_50_56%2C2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Pre Denature</td><td>95℃</td><td>2min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Elongation</td><td>72℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>10℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/18 Gelcex BAN cex cexFv VR 0.2, 3/18 Gelex J23105 VF cexFv 0.2</td><td>VF1.25</td><td>VR 1.25</td><td>12.5</td><td>9.6</td><td></td><td>25</td></tr> | ||
+ | <tr><td>3/18 Gelcex BAN clos clos Fv VR 0.2, 3/18 Gelex J23105 VF clos Rv 0.2</td><td>VF 1.25</td><td>VR 1.25</td><td>12.5</td><td>9.6</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/24 OEPCR CBDcex ctl</td><td>468.6</td><td>1.82</td><td>2.06</td></tr> | ||
+ | <tr><td>3/24 OEPCR CBDclos ctl</td><td>321.8</td><td>1.86</td><td>0.45</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/24 OEPCR BANcex ctl</td><td>77.2</td><td>1.81</td><td>1.40</td></tr> | ||
+ | <tr><td>3/24 OEPCR BAN clos ctl</td><td>23.8</td><td>1.75</td><td>1.56</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | |||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>3/24 OEPCR scFv ctl</td><td>394.2</td><td>1.83</td><td>1.19</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/24 OE PCR scFv ctl </td><td>5.1</td><td>1</td><td></td><td></td><td>1</td><td>3</td><td></td><td>19.9</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Yoshimoto --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0324" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>PCBTC3+scFv+ctl.</td><td></td><td>DH5α</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0325" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Egashira</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/24 OEPCR CBDcex ctl</td><td>4.3</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>20.7</td><td>30</td></tr> | ||
+ | <tr><td>3/24 OEPCR CBDclos ctl</td><td>6.2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>18.8</td><td>30</td></tr> | ||
+ | <tr><td>3/15 J23100+scFv C3-P2</td><td>9.7</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>15.3</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0325" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Kim</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/24 OEPCR BAN-CBDcex ctl</td><td>10</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>15</td><td>30</td></tr> | ||
+ | <tr><td>3/24 OEPCR BAN-CBDclos ctl</td><td>10</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>15</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0325" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>3/24 EPcut pSB1C3 /8</td><td>3/24 EPcut BAN-CBDcex.ctl /3.7</td><td>11.7</td><td>23.4</td></tr> | ||
+ | <tr><td>3/24 EPcut pSB1C3 /7</td><td>3/24 EPcut BAN-CBDclos.ctl /4.7</td><td>11.7</td><td>23.4</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0325" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>pSB1C3+BAN-CBDcex.ctl 3/25 ligation</td><td>-</td><td>20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>pSB1C3+BAN-CBDclos.ctl 3/25 ligation</td><td>-</td><td>20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>pSB1C3+CBDcex 3/24 ligation</td><td>-</td><td>20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>pSB1C3+CBDclos 3/24 ligation</td><td>-</td><td>20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>pSB1C3+scFv.ctl 3/24 ligation</td><td>-</td><td>20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Koide --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0328" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>BANcex ctl col.1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BAncex ctl col.2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BANclos ctl col.1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>BANclos ctl col.2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>CBDcex col.1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>CBDcex col.2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>CBDclos col,1</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>CBDclos col.2</td><td>VF 1.25 VR 1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>scFv ctl col.1</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>scFv ctl col.2</td><td></td><td></td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sukegawa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0328" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>3/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder(10)</td><td></td></tr> | ||
+ | <tr><td>2</td><td>100bb ladder(10)</td><td></td></tr> | ||
+ | <tr><td>3</td><td>BANcex ctl col.1</td><td></td></tr> | ||
+ | <tr><td>4</td><td>BANcex ctl col.2</td><td></td></tr> | ||
+ | <tr><td>5</td><td>BANclos ctl col.1</td><td></td></tr> | ||
+ | <tr><td>6</td><td>BANclos ctl col.2</td><td></td></tr> | ||
+ | <tr><td>7</td><td>CBDcex col.1</td><td></td></tr> | ||
+ | <tr><td>8</td><td>CBDcex col.2</td><td></td></tr> | ||
+ | <tr><td>9</td><td>CBDclos col.1</td><td></td></tr> | ||
+ | <tr><td>10</td><td>CBDclos col.2</td><td></td></tr> | ||
+ | <tr><td>11</td><td>scFv ctl col.1</td><td></td></tr> | ||
+ | <tr><td>12</td><td>scFv ctl col.2</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/2d/T--Kyoto--3_28_No87_Electrophoresis_Sukegawa_09_19_03_19_19.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0721" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-His-scFv glycerol stock1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-His-scFv glycerol stock2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-His-CBDcex glycerol stock1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-His-CBDcex glycerol stock2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-His-CBDclos glycerol stock1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-His-CBDclos glycerol stock2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-His-CBDcex glycerol stock1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-His-CBDcex glycerol stock2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-His-CBDclos glycerol stock1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-His-CBDclos glycerol stock2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0721" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/8 miniprep BclA-His-scFv14</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/24 miniprep BclA-His-CBDcex</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/24 miniprep BclA-His-CBDclos</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDcex</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDclos</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0722" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/4 parts awakening E0040</td><td>1</td><td>DH5α /20</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0722" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-scFv colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-scFv colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-scFv colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-scFv colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-CBDcex colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-CBDcex colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-CBDclos colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-His-CBDclos colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-CBDcex colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-CBDcex colony2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-CBDclos colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-His-CBDclos colony 2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0725" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-scFv1</td><td>183.5</td><td>1.85</td><td>2.05</td></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-scFv2</td><td>379.0</td><td>1.86</td><td>2.20</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-scFv1</td><td>189.6</td><td>1.85</td><td>2.21</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-scFv2</td><td>375.3</td><td>1.85</td><td>2.21</td></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-CBDcex1</td><td>91.9</td><td>1.87</td><td>1.90</td></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-CBDcex2</td><td>187.1</td><td>1.87</td><td>1.98</td></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-CBDclos1</td><td>41.2</td><td>1.88</td><td>2.21</td></tr> | ||
+ | <tr><td>7/22 liquid culture BclA-His-CBDclos2</td><td>109.4</td><td>1.94</td><td>2.24</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-CBDcex1</td><td>71.0</td><td>1.73</td><td>1.19</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-CBDcex2</td><td>55.5</td><td>1.89</td><td>2.23</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-CBDclos1</td><td>88.6</td><td>1.82</td><td>1.61</td></tr> | ||
+ | <tr><td>7/22 liquid culture INP-His-CBDclos2</td><td>75.8</td><td>1.86</td><td>2.17</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0725" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/25</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/25 miniprep BclA-His-scFv1</td><td>10.9</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10×L /3</td><td>0</td><td>14.1</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep BclA-His-scFv2</td><td>5.3</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H /3</td><td>3.0</td><td>16.7</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-scFv1</td><td>10.5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10×L /3</td><td>0</td><td>14.5</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep BclA-His-CBDcex2</td><td>10.7</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>14.3</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep BclA-His-CBDclos2</td><td>18.3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>6.7</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDcex1</td><td>25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDclos1</td><td>22.6</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>2.4</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Samples of "2/25 miniprep BclA-His-scFv1" and "2/25 miniprep INP-His-scFv1" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡ is restriction enzyme).</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0726" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder </td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>7/25 restriction enzyme digestion BclA-His-scFv1 DraⅡcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>7/25 restriction enzyme digestion BclA-His-scFv2 XPcut</td><td>30</td></tr> | ||
+ | <tr><td>4</td><td>7/25 restriction enzyme digestion INP-His-scFv1 DraⅡcut</td><td>30</td></tr> | ||
+ | <tr><td>5</td><td>7/25 restriction enzyme digestion BclA-His-CBDcex2 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>6</td><td>7/25 restriction enzyme digestion BclA-His-CBDclos2 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>7</td><td>7/25 restriction enzyme digestion INP-His-CBDcex1 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>8</td><td>7/25 restriction enzyme digestion INP-His-CBDclos1 SPcut</td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/af/T--Kyoto--7_26_No96_Gel_Extraction_Notsu_10_02_08_39_23.1.jpg | ||
+ | "><img src="https://static.igem.org/mediawiki/2016/1/1a/T--Kyoto-- | ||
+ | 7_26_No96_Gel_Extraction_Notsu_10_02_08_39_23.2.jpg | ||
+ | "> | ||
+ | </div> | ||
+ | |||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0726" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>7/26 Gel Extraction BclA-His-scFv2 XPcut</td><td>3.8</td><td>5.14</td><td>0.01</td></tr> | ||
+ | <tr><td>7/26 Gel Extraction BclA-His-CBDcex2 SPcut</td><td>10.4</td><td>3.24</td><td>0.01</td></tr> | ||
+ | <tr><td>7/26 Gel Extraction BclA-His-CBDclos2 SPcut</td><td>6.0</td><td>2.67</td><td>0.01</td></tr> | ||
+ | <tr><td>7/26 Gel Extraction INP-His-CBDcex1 SPcut</td><td>8.1</td><td>2.03</td><td>0.02</td></tr> | ||
+ | <tr><td>7/26 Gel Extraction INP-His-CBDclos1 SPcut</td><td>14.6</td><td>2.57</td><td>0.02</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0726" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-scFv1</td><td>10.9</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10×L /3</td><td>0</td><td>14.1</td><td>30</td></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-scFv1</td><td>3.6</td><td>1</td><td>0</td><td>0</td><td>1</td><td>10×H /3</td><td>0</td><td>3.4</td><td>10</td></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-scFv1</td><td>3.6</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6.4</td><td>10</td></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-scFv2</td><td>5.3</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H /3</td><td>3.0</td><td>16.7</td><td>30</td></tr> | ||
+ | <tr><td>/25 miniprep INP-His-scFv1</td><td>10.5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10×L /3</td><td>0</td><td>14.5</td><td>30</td></tr> | ||
+ | <tr><td>/25 miniprep INP-His-scFv1</td><td>3.5</td><td>0</td><td>0</td><td>0</td><td>1</td><td>10×H /1</td><td>0</td><td>3.5</td><td>10</td></tr> | ||
+ | <tr><td>/25 miniprep INP-His-scFv1</td><td>3.5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6.5</td><td>10</td></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-CBDcex2</td><td>10.7</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>14.3</td><td>30</td></tr> | ||
+ | <tr><td>7/25 miniprep BclA-His-CBDclos2</td><td>18.3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>6.7</td><td>30</td></tr> | ||
+ | <tr><td>7/25 miniprep INP-His-CBDcex2</td><td>25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>7/25 miniprep INP-His-CBDclos2</td><td>25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Samples of "2/25 miniprep BclA-His-scFv(10.9µl)" and "2/25 miniprep INP-His-scFv1(10.5µl)" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡ is restriction enzyme).</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0726" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>2/26Restriction Enzyme Digestion BclA-His-scFv1 DraⅡcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>2/26Restriction Enzyme Digestion BclA-His-scFv1 XPcut</td><td>10</td></tr> | ||
+ | <tr><td>4</td><td>2/26Restriction Enzyme Digestion BclA-His-scFv1</td><td>10</td></tr> | ||
+ | <tr><td>5</td><td>2/26Restriction Enzyme Digestion INP-His-scFv1 DraⅡcut</td><td>30</td></tr> | ||
+ | <tr><td>6</td><td>2/26Restriction Enzyme Digestion INP-His-scFv1EPcut</td><td>10</td></tr> | ||
+ | <tr><td>7</td><td>2/26Restriction Enzyme Digestion INP-His-scFv1 </td><td>10</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>1'</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2'</td><td>2/26Restriction Enzyme Digestion BclA-His-scFv2 XPcut</td><td>30</td></tr> | ||
+ | <tr><td>3'</td><td>2/26Restriction Enzyme Digestion BclA-His-CBDcex2 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>4'</td><td>2/26Restriction Enzyme Digestion BclA-His-CBDclos2 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>5'</td><td>2/26Restriction Enzyme Digestion INP-His-CBDcex2 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>6'</td><td>2/26Restriction Enzyme Digestion INP-His-CBDclos2 SPcut</td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/59/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-1.jpg | ||
+ | "><img src="https://static.igem.org/mediawiki/2016/3/36/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-2.jpg"><img src="https://static.igem.org/mediawiki/2016/a/a1/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.2-1.jpg | ||
+ | "><img src="https://static.igem.org/mediawiki/2016/f/fe/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.2-2.jpg | ||
+ | "> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0727" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert DNA(7/27purification) /µ?</th><th>Vector DNA(7/25purification) /µ?</th><th>Ligation High /µ?</th><th>Total</th></tr> | ||
+ | <tr><td>-</td><td>BclA-scFv1 DraⅡcut /7.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>-</td><td>INP-scFv1 DraⅡcut /7.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>BclAscFv2 XPcut /7.16</td><td>BclA-CBDcex2 SPcut /0.34</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>BclAscFv2 XPcut /3.75</td><td>BclA-CBDclos2 SPcut /3.75</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>BclAscFv2 XPcut /1.88</td><td>INP-CBDcex2 SPcut /5.62</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>BclAscFv2 XPcut /5.0</td><td>INP-CBDclos2 SPcut /2.5</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0727" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>BclAscFv1 DraⅡcut</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INPscFv1 DraⅡcut</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclAscFv+BclACBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclAscFv+BclACBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclAscFv+INPCBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclAscfv+INPCBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0727" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>7/27 Gel Extraction BclA-His-scFv1 DraⅡcut</td><td>4.8</td><td>-7.11</td><td>1.04</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction INP-His-scFv1 DraⅡcut</td><td>40.4</td><td>1.32</td><td>1.00</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction BclA-His-scFv2 XPcut</td><td>9.3</td><td>-4.24</td><td>0.02</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction BclA-His-CBDcex2 SPcut</td><td>174.5</td><td>1.32</td><td>0.84</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction BclA-His-CBDclos2 SPcut</td><td>9.7</td><td>5.84</td><td>0.19</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction INP-His-CBDcex2 SPcut</td><td>3.7</td><td>-2.82</td><td>0.14</td></tr> | ||
+ | <tr><td>7/27 Gel Extraction INP-His-CBDclos2 SPcut</td><td>22.6</td><td>2.51</td><td>0.21</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA /µ?</th><th>EcoR1 /µ?</th><th>Xba1 /µ?</th><th>Spe1 /µ?</th><th>Pst1 /µ?</th><th>DraⅡ /µ?</th><th>Buffer /µ?</th><th>BSA /µ?</th><th>MilliQ /µ?</th><th>Total /µ?</th></tr> | ||
+ | <tr><td>BclAscFv2 /5.3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>10×L /3</td><td>0</td><td>20.7</td><td>30</td></tr> | ||
+ | <tr><td>BclAscFv2 /5.3</td><td>0</td><td>1</td><td>0</td><td>1</td><td>0</td><td>10×H /3</td><td>3</td><td>16.7</td><td>30</td></tr> | ||
+ | <tr><td>INPscFv2 /5.3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>10×L /3</td><td>0</td><td>20.7</td><td>30</td></tr> | ||
+ | <tr><td>BclAcex2 /10.7</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>10×M /3</td><td>0</td><td>14.3</td><td>30</td></tr> | ||
+ | <tr><td>BclAclos1 /25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>INPcex1,2 /25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>INPclos1,2 /25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>10×M /3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td></td><td>7/28 BclA-scFv2 XPcut</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">0.5µg/µl<br>260/280 2.36<br>260/230 1.15</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Kaneko</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/22 scFv colony2 </td><td>2</td><td>DH5α /20</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert DNA /(µl)</th><th>Vector DNA /(µl)</th><th>Ligation high /(µl)</th><th>Total /(µl)</th></tr> | ||
+ | <tr><td>-</td><td>7/28 Restriction Enzyme BclA-scFv DraⅡcut /7.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>-</td><td>7/28 Restriction Enzyme INP-scFv DraⅡcut /7.5</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>7/28 ligation BclA-scFv control</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/28 ligation INP-scFv control</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/22 miniprep BAN-scFv</td><td>-</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0728" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>7/28</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-scFv glycerol stock </td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-scFv colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-CBDcex colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation BclA-CBDclos colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-CBDcex colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation INP-CBDclos colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/21 transformation </td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0801" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>7/28 transformaton BclA control colony4</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>7/28 transformaton BclA control colony5</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>7/28 transformaton BclA control colony6</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>7/28 transformaton INP control colony4</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>7/28 transformaton INP control colony5</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>7/28 transformaton INP control colony6</td><td>VF/1.25 VR/1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0801" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>1min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>10sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elogation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0801" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>7/28 liquid culture BclA-scFv</td><td>239.1</td><td>1.87</td><td>1.78</td></tr> | ||
+ | <tr><td>7/28 liquid culture INP-scFv colony1</td><td>263.4</td><td>1.86</td><td>1.99</td></tr> | ||
+ | <tr><td>7/28 liquid culture E0040 colony1</td><td>98.8</td><td>1.84</td><td>2.15</td></tr> | ||
+ | <tr><td>7/28 liquid culture BclA-CBDcex colony1</td><td>109.9</td><td>1.82</td><td>1.37</td></tr> | ||
+ | <tr><td>7/28 liquid culture BclA-CBDclos colony1</td><td>94.1</td><td>1.89</td><td>1.93</td></tr> | ||
+ | <tr><td>7/28 liquid culture INP-CBDcex colony1</td><td>111.4</td><td>1.82</td><td>1.39</td></tr> | ||
+ | <tr><td>7/28 liquid culture INP-CBDclos colony1</td><td>185.1</td><td>1.81</td><td>1.51</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0801" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>8/1 colonyPCR BclA control colony4 /10</td><td>672</td></tr> | ||
+ | <tr><td>2</td><td>8/1 colonyPCR BclA control colony5 /10</td><td>672</td></tr> | ||
+ | <tr><td>3</td><td>8/1 colonyPCR BclA control colony6 /10</td><td>672</td></tr> | ||
+ | <tr><td>4</td><td>8/1 colonyPCR INP control colony4 /10</td><td>1563</td></tr> | ||
+ | <tr><td>5</td><td>8/1 colonyPCR INP control colony5 /10</td><td>1563</td></tr> | ||
+ | <tr><td>6</td><td>8/1 colonyPCR INP control colony6 /10</td><td>1563</td></tr> | ||
+ | <tr><td>7</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/83/T--Kyoto--8_1_No12_Electrophoresis_Notsu_09_30_08_29_26.1.jpg"><img src="https://static.igem.org/mediawiki/2016/c/c9/T--Kyoto--8_1_No12_Electrophoresis_Notsu_09_30_08_29_26.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0801" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/1 miniprep BclA-scFv</td><td>8.36</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H 3</td><td>3</td><td>13.64</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep BclA-CBDcex colony1</td><td>18.2</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×H /3</td><td>0</td><td>6.8</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep BclA-CBDclos colony1</td><td>21.3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>3.7</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep INP-CBDcex colony1</td><td>18.0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>7.0</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep INP-CBDclos colony1</td><td>10.8</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>16.2</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0802" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>BclA-scFv XPcut</td><td>30</td></tr> | ||
+ | <tr><td>2</td><td>BclA-CBDcex SPcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>BclA-CBDclos SPcut</td><td>30</td></tr> | ||
+ | <tr><td>4</td><td>INP-CBDcex SPcut</td><td>30</td></tr> | ||
+ | <tr><td>5</td><td>INP-CBDclos SPcut</td><td>30</td></tr> | ||
+ | <tr><td>6</td><td>1kb ladder </td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/28/T--Kyoto--8_2_No14_Gel_Extraction_Notsu_09_30_08_40_44.1.jpg"><img src="https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--8_2_No14_Gel_Extraction_Notsu_09_30_08_40_44.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0802" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>Concentration ng/µl</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/2 Electrophoresis BclA-scFv XPcut</td><td>1.0</td><td>23.20</td><td>0.12</td></tr> | ||
+ | <tr><td>8/2 Electrophoresis BclA-CBDcex SPcut</td><td>10.8</td><td>1.89</td><td>1.35</td></tr> | ||
+ | <tr><td>8/2 Electrophoresis BclA-CBDclos SPcut</td><td>18.0</td><td>1.92</td><td>1.88</td></tr> | ||
+ | <tr><td>8/2 Electrophoresis INP-CBDcex SPcut</td><td>25.4</td><td>1.93</td><td>1.81</td></tr> | ||
+ | <tr><td>8/2 Electrophoresis INP-CBDclos SPcut</td><td>60.9</td><td>1.23</td><td>1.05</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0802" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/2 purification BclA-CBDcex SPcut /3.5</td><td>8/2 purification BclA-scFv XPcut /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/2 purification BclA-CBDclos SPcut /3.5</td><td>8/2 purification BclA-scFv XPcut /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/2 purification INP-CBDcex SPcut /3.5</td><td>8/2 purification BclA-scFv XPcut /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/2 purification INP-CBDclos SPcut /3.5</td><td>8/2 purification BclA-scFv XPcut /4</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0802" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/2 ligation BclA-scFv+BclA-CBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/2 ligation BclA-scFv+BclA-CBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/2 ligation BclA-scFv+INP-CBDcex</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/2 ligation BclA-scFv+INP-CBDclos</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0802" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/2</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-CBDcex glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-CBDclos glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDcex glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDclos glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0803" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>7/28 transformation BclA control colony5</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/28 transformation BclA control colony6</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/28 transformation INP control colony4</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/28 transformation INP control colony5</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>7/28 transformation INP control colony6</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0803" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/1 miniprep BclA-scFv</td><td>8.36</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H /3</td><td>3</td><td>13.64</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep BclA-CBDcex</td><td>18.2</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×H /3</td><td>0</td><td>6.8</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep BclA-CBDclos</td><td>21.3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>3.7</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep INP-CBDcex </td><td>18.0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>7.0</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep INP-CBDclos</td><td>10.8</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>16.2</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0803" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Vector/(µl)</th><th>Insert/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/1 Restriction Enzyme Digestion BclA-CBDcex /3.5</td><td>8/1 Restriction Enzyme Digestion BclA-scFv /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/1 Restriction Enzyme Digestion BclA-CBDclos /3.5</td><td>8/1 Restriction Enzyme Digestion BclA-scFv /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/1 Restriction Enzyme Digestion INP-CBDcex /3.5</td><td>8/1 Restriction Enzyme Digestion BclA-scFv /4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/1 Restriction Enzyme Digestion INP-CBDclos /3.5</td><td>8/1 Restriction Enzyme Digestion BclA-scFv /4</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0803" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/3</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/3 ligation BclA-scFv+BclA-CBDcex</td><td>15</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/3 ligation BclA-scFv+BclA-CBDclos</td><td>15</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/3 ligation BclA-scFv+INP-CBDcex</td><td>15</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/3 ligation BclA-scFv+INP-CBDclos</td><td>15</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDcex colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDcex colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDcex colony3</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDcex colony4</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDclos colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDclos colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDclos colony3</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+BclA-CBDclos colony4</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDcex colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDcex colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDcex colony3</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDcex colony4</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDclos colony1</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDclos colony2</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDclos colony3</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/3 transformation BclA-scFv+INP-CBDclos colony4</td><td>VF 1.25 VR1.25</td><td>10</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>1min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>95℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>10sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>4℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/3 liquid culture BclA control colony5</td><td>137.2</td><td>1.77</td><td>1.03</td></tr> | ||
+ | <tr><td>8/3 liquid culture BclA control colony6</td><td>117.6</td><td>1.87</td><td>1.54</td></tr> | ||
+ | <tr><td>8/3 liquid culture INP control4</td><td>165.2</td><td>1.87</td><td>1.68</td></tr> | ||
+ | <tr><td>8/3 liquid culture INP control5</td><td>150.6</td><td>1.90</td><td>1.81</td></tr> | ||
+ | <tr><td>8/3 liquid culture INP control6</td><td>147.4</td><td>1.79</td><td>1.63</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Sequence</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>No</th><th>primer(10µM)</th><th>MilliQ</th><th>Total</th></tr> | ||
+ | <tr><td>1 / 2</td><td>BclA_5_VF(2.18µl) / BclA_5_VR(2.18µl)</td><td>5.82µl / 5.82µl</td><td>10µl</td></tr> | ||
+ | <tr><td>3 / 4</td><td>BclA_6_VF(2.55µl) / BclA_6_VR(2.55µl)</td><td>5.45µl / 5.45µl</td><td>10µl</td></tr> | ||
+ | <tr><td>5 / 6</td><td>INP_4_VF(1.81µl) / INP_4_VR(1.81µl)</td><td>6.19µl / 6.19µl</td><td>10µl</td></tr> | ||
+ | <tr><td>7 / 8</td><td>INP_5_VF(1.99µl) / INP_5_VR(1.99µl)</td><td>6.01µl / 6.01µl</td><td>10µl</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>8/4 colony PCR BclA-scFv+BclA-CBDcex colony1 /10</td><td>1716</td></tr> | ||
+ | <tr><td>2</td><td>8/4 colony PCR BclA-scFv+BclA-CBDcex colony2 /10</td><td>1716</td></tr> | ||
+ | <tr><td>3</td><td>8/4 colony PCR BclA-scFv+BclA-CBDcex colony3 /10</td><td>1716</td></tr> | ||
+ | <tr><td>4</td><td>8/4 colony PCR BclA-scFv+BclA-CBDcex colony4 /10</td><td>1716</td></tr> | ||
+ | <tr><td>5</td><td>8/4 colony PCR BclA-scFv+BclA-CBDclos colony1 /10</td><td>1716</td></tr> | ||
+ | <tr><td>6</td><td>8/4 colony PCR BclA-scFv+BclA-CBDclos colony2 /10</td><td>1716</td></tr> | ||
+ | <tr><td>7</td><td>8/4 colony PCR BclA-scFv+BclA-CBDclos colony3 /10</td><td>1716</td></tr> | ||
+ | <tr><td>8</td><td>8/4 colony PCR BclA-scFv+BclA-CBDclos colony4 /10</td><td>1716</td></tr> | ||
+ | <tr><td>9</td><td>100bp ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>10</td><td>1kb ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>11</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>12</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/7f/T--Kyoto--8_4_No26_Electrophoresis_Notsu_10_01_11_15_31.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0804" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>8/4 colony PCR BclA-scFv+INP-CBDcex colony1 /10</td><td>2606</td></tr> | ||
+ | <tr><td>2</td><td>8/4 colony PCR BclA-scFv+INP-CBDcex colony2 /10</td><td>2606</td></tr> | ||
+ | <tr><td>3</td><td>8/4 colony PCR BclA-scFv+INP-CBDcex colony3 /10</td><td>2606</td></tr> | ||
+ | <tr><td>4</td><td>8/4 colony PCR BclA-scFv+INP-CBDcex colony4 /10</td><td>2606</td></tr> | ||
+ | <tr><td>5</td><td>8/4 colony PCR BclA-scFv+INP-CBDclos colony1 /10</td><td>2606</td></tr> | ||
+ | <tr><td>6</td><td>8/4 colony PCR BclA-scFv+INP-CBDclos colony2 /10</td><td>2606</td></tr> | ||
+ | <tr><td>7</td><td>8/4 colony PCR BclA-scFv+INP-CBDclos colony3 /10</td><td>2606</td></tr> | ||
+ | <tr><td>8</td><td>8/4 colony PCR BclA-scFv+INP-CBDclos colony4 /10</td><td>2606</td></tr> | ||
+ | <tr><td>9</td><td>100bp ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>10</td><td>1kb ladder /5</td><td>-</td></tr> | ||
+ | <tr><td>11</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>12</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/cc/T--Kyoto--8_4_No27_Electrophoresis_Notsu_10_01_11_18_45.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0805" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/5</h3></span> | ||
+ | <span class="keln_exp"><h4>PartsAwaking</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Description</th><th>PartName</th><th>Backbone</th><th>Well</th><th>PlateYear</th><th>PlateNumber</th><th>MilliQ(µl)</th></tr> | ||
+ | <tr><td>RFP</td><td>J23105</td><td>J61002</td><td>17N</td><td>2016</td><td>4</td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0805" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/5 Parts awakening J23105</td><td>1µl</td><td>DH5α /20</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0809" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Iguchi, Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/1 miniprep BclA-scFv</td><td>8.36</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H /3</td><td>3</td><td>13.69</td><td>30</td></tr> | ||
+ | <tr><td>8/1 and 7/25 miniprep BclA-CBDcex</td><td>18.</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×H /3</td><td>0</td><td>6.8</td><td>30</td></tr> | ||
+ | <tr><td>2/25 miniprep BclA-CBDclos</td><td>21.3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>3.7</td><td>30</td></tr> | ||
+ | <tr><td>2/25 and 7/25 minniprep INP-CBDcex</td><td>18.0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>7.0</td><td>30</td></tr> | ||
+ | <tr><td>8/1 miniprep INP-CBDclos</td><td>10.8</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M /3</td><td>0</td><td>16.2</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0809" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/1 transformation J23105</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0809" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/3 liquid culture BclA-CBDccex</td><td>225.8</td><td>1.74</td><td>1.63</td></tr> | ||
+ | <tr><td>8/3 liquid culture BclA-CBDclos</td><td>102.3</td><td>1.80</td><td>1.66</td></tr> | ||
+ | <tr><td>8/3 liquid culture INP-CBDcex</td><td>112.4</td><td>1.86</td><td>1.77</td></tr> | ||
+ | <tr><td>8/3 liquid culture INP-CBDclos</td><td>42.8</td><td>1.76</td><td>0.78</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0810" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>A1</td><td>1kb ladder</td><td>10</td></tr> | ||
+ | <tr><td>A2</td><td>BclA-scFv 8/9XPcut</td><td>30</td></tr> | ||
+ | <tr><td>A3</td><td>BclA-CBDcex 8/9 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>A4</td><td>BclA-CBDclos 8/9 SPcu</td><td>30</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>-</td></tr> | ||
+ | <tr><td>B1</td><td>1kb ladder</td><td>10</td></tr> | ||
+ | <tr><td>B2</td><td>INP-CBDcex 8/9 SPcut</td><td>30</td></tr> | ||
+ | <tr><td>B3</td><td>INP-CBDclos 8/9 SPcut</td><td>30</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/be/T--Kyoto--8_10_No31_Gel_Extraction_Notsu_10_01_11_59_14.1.jpg"><img src="8_10_No31_Gel_Extraction_Notsu_10_01_11_59_14.2.jpg | ||
+ | https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0810" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>BclA-scFv 8/9XPcut</td><td>2.6</td><td>1.13</td><td>0.58</td></tr> | ||
+ | <tr><td>BclA-CBDcex 8/9 SPcut</td><td>6.3</td><td>1.54</td><td>0.97</td></tr> | ||
+ | <tr><td>BclA-CBDclos 8/9 SPcut</td><td>4.7</td><td>1.49</td><td>0.48</td></tr> | ||
+ | <tr><td>INP-CBDcex 8/9 SPcut</td><td>4.4</td><td>1.39</td><td>0.67</td></tr> | ||
+ | <tr><td>INP-CBDclos 8/9 SPcut</td><td>3.5</td><td>1.29</td><td>0.77</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0810" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/10</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/9 liquid culture J23105 (AMP) RFP colony3</td><td>178.8</td><td>1.98</td><td>1.92</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0812" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/2 liquid culture INP-CBDclos</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv glycerol stock 1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv glycerol stock 2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv glycerol stock 3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv glycerol stock 4</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0818" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Notsu</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/12 liquid culture INP-CBDclos</td><td>45.3</td><td>1.82</td><td>1.31</td></tr> | ||
+ | <tr><td>8/12 liquid culture BclA-scFv glycerol stock 1</td><td>101.6</td><td>1.59</td><td>0.71</td></tr> | ||
+ | <tr><td>8/12 liquid culture BclA-scFv glycerol stock 2</td><td>100.7</td><td>1.67</td><td>0.78</td></tr> | ||
+ | <tr><td>8/12 liquid culture BclA-scFv glycerol stock 3</td><td>77.8</td><td>1.60</td><td>0.70</td></tr> | ||
+ | <tr><td>8/12 liquid culture BclA-scFv glycerol stock 4</td><td>41.0</td><td>1.87</td><td>1.27</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0820" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-CBDcex</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-CBDclos</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDcex</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDclos</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0821" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-scFv [1](glisto)</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv [2]</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv [3]</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-scFv [4]</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion 1.1kb ladder</td><td>5</td><td></td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion Bcla-cex</td><td>25</td><td>2520</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion Bcla-clos</td><td>25</td><td>2520</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion INP-cex(s,pcut)</td><td>25</td><td>3500</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion INP-clos</td><td>25</td><td>3500</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion INP-cex</td><td>25</td><td>2541</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion J23105 (AMP) RFP 8/21 old</td><td>25</td><td>950</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion Liquid culture Bcla-scfv glisto 4</td><td>25</td><td>932</td></tr> | ||
+ | <tr><td>8/22 Restraction Ensyme Digestion Bcla-scfv glisto 2</td><td>25</td><td>932</td></tr> | ||
+ | <tr><td>100ladder</td><td>5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.1.jpg"><img src="https://static.igem.org/mediawiki/2016/a/a4/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.2.jpg"><img src="https://static.igem.org/mediawiki/2016/f/fa/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.3.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/12 Liquid cultureBclA-scFv glisto [4]</td><td>38</td><td>0</td><td>1</td><td>0</td><td>1</td><td>5</td><td>5</td><td>0</td><td>50</td></tr> | ||
+ | <tr><td>8/12 Liquid cultureBclA-scFv glisto [2]</td><td>38</td><td>0</td><td>1</td><td>0</td><td>1</td><td>5</td><td>5</td><td>0</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yoshimoto, Ohara, Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA control</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP control</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/9 J23105 RFP</td><td></td><td>DH5α /20</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 1st</h4></span> | ||
+ | <span class="keln_researcher">Yoshimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAclos+</td><td>0.208/0.206/0.206</td><td>0.131/0.135/0.14</td><td>0.111/0.112/0.109</td></tr> | ||
+ | <tr><td>INPclos+</td><td>0.183/0.186/0.184</td><td>0.239/0.23/0.22</td><td>0.158/0.155/0.169</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0822" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/22</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 2nd</h4></span> | ||
+ | <span class="keln_researcher">Yoshimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAclos+</td><td>0.208/0.205/0.204</td><td>0.078/0.074/0.075</td><td>0.056/0.056/0.039</td></tr> | ||
+ | <tr><td>INPclos+</td><td>0.183/0.181/0.187</td><td>0.093/0.097/0.099</td><td>0.067/0.073/0.069</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0823" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/23</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 3rd</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (15min)</td><td>0.247/0.247/0.248</td><td>0.221/0.224/0.218</td><td>0.085/0.075/0.073</td></tr> | ||
+ | <tr><td>BclAcex+ (30min)</td><td>0.247/0.247/0.248</td><td>0.243/0.229/0.228</td><td>0.034/0.048/0.038</td></tr> | ||
+ | <tr><td>BclAcex+ (60min)</td><td>0.247/0.247/0.248</td><td>0.224/0.221/0.217</td><td>0.065/0.064/0.065</td></tr> | ||
+ | <tr><td>BclAcex- (15min)</td><td>0.247/0.247/0.248</td><td>0.257/0.253/0.258</td><td></td></tr> | ||
+ | <tr><td>BclAcex- (30min)</td><td>0.247/0.247/0.248</td><td>0.236/0.230/0.235</td><td></td></tr> | ||
+ | <tr><td>BclAcex- (60min)</td><td>0.247/0.247/0.248</td><td>0.249/0.248/0.247</td><td></td></tr> | ||
+ | <tr><td> cellulose only (60min)</td><td></td><td>0.021/0.021/0.023</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0823" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/23</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 4th</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (2ml tube)</td><td>0.227/0.226/0.225</td><td>0.108/0.108/0.108</td><td>0.076/0.074/0.081</td></tr> | ||
+ | <tr><td>BclActl+ (2ml tube)</td><td>0.186/0.203/0.189</td><td>0.082/0.081/0.082</td><td>0.055/0.046/0.044</td></tr> | ||
+ | <tr><td>BclAcex- (2ml tube)</td><td>0.235/0.237/0.233</td><td>0.261/0.262/0.254</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (1.5ml tube)</td><td>0.237/0.226/0.236</td><td>0.159/0.157/0.158</td><td>0.066/0.063/0.065</td></tr> | ||
+ | <tr><td>BclActl+ (1.5ml tube)</td><td>0.177/0.173/0.179</td><td>0.112/0.109/0.113</td><td>0.080/0.076/0.095</td></tr> | ||
+ | <tr><td>BclAcex- (1.5ml tube)</td><td>0.240/0.235/0.238</td><td>0.255/0.254/0.253</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/5 transormation J2305</td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>8/24 BclA-scFv 1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/24 BclA-scFv 2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/24 BclA-scFv 3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/24 BclA-scFv 4</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>1. 8/23Liquidculture Bcla scFv</td><td>111.6</td><td>1.98</td><td>1.82</td></tr> | ||
+ | <tr><td> 2. 8/23Liquidculture Bcla scFv </td><td>143.1</td><td>2.00</td><td>2.03</td></tr> | ||
+ | <tr><td> 2'. 8/23Liquidculture Bcla scFv</td><td>141.6</td><td>2.04</td><td>2.10</td></tr> | ||
+ | <tr><td> 3. 8/23Liquidculture Bcla scFv</td><td>145.2</td><td>2.07</td><td>2.01</td></tr> | ||
+ | <tr><td>8/23Liquidculture J23105</td><td>63.1</td><td>2.01</td><td>1.69</td></tr> | ||
+ | <tr><td>8/23Liquidculture J23105</td><td>68.1</td><td>2.00</td><td>1.75</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 5th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>INPcex+</td><td>0.195/0.191/0.192</td><td>0.103/0.102/0.109</td><td>0.059/0.051/0.055</td></tr> | ||
+ | <tr><td>INPcex-</td><td>0.190/0.191/0.205</td><td>0.186/0.187/0.186</td><td></td></tr> | ||
+ | <tr><td>INPclos+</td><td>0.187/0.187/0.188</td><td>0.121/0.122/0.126</td><td>0.051/0.058/0.059</td></tr> | ||
+ | <tr><td>INPclos-</td><td>0.196/0.195/0.191</td><td>0.206/0.204/0.202</td><td></td></tr> | ||
+ | <tr><td>INPctl+</td><td>0.172/0.163/0.162</td><td>0.116/0.177/0.124</td><td>0.066/0.063/0.062</td></tr> | ||
+ | <tr><td>INPctl-</td><td>0.171/0.175/0.165</td><td>0.167/0.182/0.171</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0824" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/24</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 6th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>INPcex+</td><td>0.184/0.184/0.189</td><td>0.104/0.106/0.106</td><td>0.044/0.044/0.056</td></tr> | ||
+ | <tr><td>INPcex-</td><td>0.186/0.190/0.184</td><td>0.182/0.181/0.184</td><td></td></tr> | ||
+ | <tr><td>INPclos+</td><td>0.184/0.189/0.185</td><td>0.097/0.098/0.099</td><td>0.037/0.037/0.038</td></tr> | ||
+ | <tr><td>INPclos-</td><td>0.187/0.192/0.190</td><td>0.185/0.184/0.189</td><td></td></tr> | ||
+ | <tr><td>INPctl+</td><td>0.173/0.184/0.180</td><td>0.095/0.099/0.095</td><td>0.045/0.046/0.046</td></tr> | ||
+ | <tr><td>INPctl-</td><td>0.185/0.184/0.183</td><td>0.175/0.175/0.175</td><td></td></tr> | ||
+ | <tr><td>BclAcex+</td><td>0.170/0.168/0.174</td><td>0.099/0.094/0.092</td><td>0.037/0.032/0.036</td></tr> | ||
+ | <tr><td>BclAcex-</td><td>0.186/0.189/0.171</td><td>0.164/0.167/0.164</td><td></td></tr> | ||
+ | <tr><td>cellulose only</td><td>0.006/0.005/0.006</td><td>0.013/0.010/0.006</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0825" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/25</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 7th</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+</td><td>0.181/0.181/0.181</td><td>0.192/0.196/0.195</td><td>0/0/0</td></tr> | ||
+ | <tr><td>BclAcex-</td><td>0.149/0.150/0.151</td><td>0.221/0.213/0.211</td><td></td></tr> | ||
+ | <tr><td>BclActl+</td><td>0.169/0.165/0.167</td><td>0.154/0.156/0.157</td><td>0.012/0.013/0.011</td></tr> | ||
+ | <tr><td>BclActl-</td><td>0.151/0.153/0.154</td><td>0.187/0.178/0.173</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/10 miniprep J23105 /3</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>7</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/24 OD0.206 INP-CBDclos /3</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>7</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | <tr><td>8/24 OD0.202 INP-CBDcex</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>7</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>8/26 PCR INP-CBDcex VFVR /15</td><td></td></tr> | ||
+ | <tr><td>3</td><td>8/26 PCR INP-CBDclos VFVR /15</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4b/T--Kyoto--8_26_No62_Electrophoresis_Notsu_10_01_02_14_38.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>8/26 Gel Extraction Bcla cex</td><td>8.3</td><td>1.52</td><td>0.25</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction Bcla clos</td><td>10.2</td><td>1.70</td><td>0.26</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction INP cex</td><td>11.4</td><td>1.76</td><td>0.15</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction INP clos</td><td>7.7</td><td>1.62</td><td>0.09</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction Bcla scFv glisto</td><td>7.0</td><td>1.59</td><td>0.20</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction Bcla scFv glisto</td><td>14.1</td><td>6.33</td><td>0.04</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 8th</h4></span> | ||
+ | <span class="keln_researcher">Yoshimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+</td><td>0.183/0.185/0.188</td><td>0.116/0.115/0.114</td><td>0.086/0.086/0.089</td></tr> | ||
+ | <tr><td>BclActl+</td><td>0.116/0.115/0.114</td><td>0.096/0.092/0.087</td><td>0.09/0.079/0.084</td></tr> | ||
+ | <tr><td>cellulose only</td><td></td><td>0.037/0.035/0.037</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0826" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/26</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 9th</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+</td><td>0.186/0.186/0.184</td><td>0.170/0.169/0.170</td><td>0.021/0.024/0.020</td></tr> | ||
+ | <tr><td>BclAcex-</td><td>0.154/0.149/0.149</td><td>0.142/0.143/0.141</td><td></td></tr> | ||
+ | <tr><td>BclActl+</td><td>0.194/0.189/0.189</td><td>0.167/0.164/0.159</td><td>0.010/0.009/0.008</td></tr> | ||
+ | <tr><td>BclActl-</td><td>0.162/0.168/0.169</td><td>0.159/0.162/0.165</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0829" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert DNA</th><th>vector DNA</th><th>Ligation High</th><th>Total</th></tr> | ||
+ | <tr><td>8/26 Gel Extraction BclAscFv /3.4</td><td>8/26 Gel Extraction Bcla CBDcex /4.1</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction BclAscFv /3.1</td><td>8/26 Gel Extraction Bcla CBD clos /4.4</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction BclAscFvBclAscFv /3.8</td><td>8/26 Gel Extraction Bcla CBD cex /4.7</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>8/26 Gel Extraction BclAscFv /2.6</td><td>8/26 Gel Extraction Bcla CBD clos /4.9</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0829" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl</td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 0.2µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.1µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.02µl</td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/0e/T--Kyoto--8_29_No68_General_Miyazaki_10_17_05_42_18.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">blotting CC<br>1'antibody time/buffer 1:3000 6ml 1h<br>2'antibody time/buffer 1:10000 10ml 1h<br>Blocking 1day<br>transfer 100mA 1h</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0829" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>BclA-CBDcex</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>10</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | <tr><td>BclA-CBDclos</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>10</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-CBDcex</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>10</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-CBDclos</td><td>VF /1.25</td><td>VR /1.25</td><td>-</td><td>10</td><td>KAPA2GFAST /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--8_29_No69_PCR_%28Target%29_Notsu_10_01_02_11_53.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0829" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>ligation</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert DNA</th><th>Vector DNA</th><th>Ligation High </th><th>Total</th></tr> | ||
+ | <tr><td>BclAscFv XPcut 6.3</td><td>INPCBDcex SPcut 1.2</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>BclAscFv XPcut 6.4</td><td>INPCBDclos SPcut 1.1</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0829" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>8/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>BclA scFv 1</td><td>8.4</td><td>1.63</td><td>0.11</td></tr> | ||
+ | <tr><td>BclA scFv 2</td><td>5.6</td><td>1.91</td><td>0.04</td></tr> | ||
+ | <tr><td>INP CBDcex</td><td>44.8</td><td>1.90</td><td>0.70</td></tr> | ||
+ | <tr><td>INP CBDclos</td><td>33.1</td><td>1.82</td><td>0.68</td></tr> | ||
+ | <tr><td>BclA cex</td><td>26.3</td><td>1.93</td><td>0.62</td></tr> | ||
+ | <tr><td>BclA clos </td><td>24.4</td><td>1.93</td><td>0.66</td></tr> | ||
+ | <tr><td>J23105 VF VR</td><td>33.2</td><td>1.88</td><td>0.24</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/10 J23105 RFP miniprep </td><td>20</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H 3</td><td>3</td><td>2</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/29 Ligation Bclascfv-BclaCBDcex</td><td>2</td><td>CH5a/20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃Bclascfv-BclaCBDclos</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃Bclascfv-INPCBDcex</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃Bclascfv-INPCBDclos</td><td>2</td><td>〃</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>8/20 SPcut Bcla-CBDcex/25</td><td>2520</td></tr> | ||
+ | <tr><td>2</td><td>〃Bcla-CBDclos/25</td><td>〃</td></tr> | ||
+ | <tr><td>3</td><td>〃INP-CBDcex/25</td><td>〃</td></tr> | ||
+ | <tr><td>4</td><td>〃INP-CBDclos/25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--9_5_No74_Electrophoresis_Yamamoto_09_30_10_51_18.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>100bp ladder/5</td><td>6×loading Dye -</td></tr> | ||
+ | <tr><td>2</td><td>8/10 miniprep 9/5 xpcut J23105RFP/30</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-CBDcex /20ml</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-control /20ml</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0905" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>9/5 electrophoresis 2 </td><td>J23105 RFP XPcut backbone</td><td></td></tr> | ||
+ | <tr><td>9/5 electrophoresis 2</td><td>J23105 RFP XPcut insert</td><td></td></tr> | ||
+ | <tr><td>9/5 electrophoresis 1</td><td>BclAcex SPcut</td><td></td></tr> | ||
+ | <tr><td>9/5 electrophoresis 2</td><td>BclAclos SPcut</td><td></td></tr> | ||
+ | <tr><td>9/5 electrophoresis 3</td><td>INPcex SPcut</td><td></td></tr> | ||
+ | <tr><td>9/5 electrophoresis 4</td><td>INPclos SPcut</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>Bclascfv+BclaCBDcex colony1</td><td>vf1.25 vr1.25</td><td>10</td><td>KAPA2GFAST 12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony4</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>Bclascfv+BclaCBDclos colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony4</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>Bclascfv+INPCBDcex colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony4</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>Bclascfv+INPCBDclos colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony2</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony3</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony4</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert DNA(µl)</th><th>Vector DNA/(µl)</th><th>Ligation High/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/5 Gel Extraction BclA cex SPcut/7</td><td>9/5 Gel Extraction J23105 xpcut 36/0.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>9/5 Gel Extraction BclA clos SPcut/7</td><td>9/5 Gel Extraction J23105 xpcut 36/0.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>9/5 Gel Extraction INP cex SPcut/7</td><td>9/5 Gel Extraction J23105 xpcut 36/0.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>9/5 Gel Extraction INP clos SPcut/7</td><td>9/5 Gel Extraction J23105 xpcut 36/0.5</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/5</td><td>0</td></tr> | ||
+ | <tr><td>2</td><td>9/6 PCR scfv+Bclacex colony1/15</td><td>0</td></tr> | ||
+ | <tr><td>3</td><td>〃2/15</td><td>0</td></tr> | ||
+ | <tr><td>4</td><td>〃3/15</td><td>0</td></tr> | ||
+ | <tr><td>5</td><td>〃4/15</td><td>0</td></tr> | ||
+ | <tr><td>6</td><td>9/6 PCR scfv+Bclaclos colony1</td><td>0</td></tr> | ||
+ | <tr><td>7</td><td>〃2</td><td>0</td></tr> | ||
+ | <tr><td>8</td><td>〃3</td><td>0</td></tr> | ||
+ | <tr><td>9</td><td>〃4</td><td>0</td></tr> | ||
+ | <tr><td>10</td><td>9/6 PCR scfv+INPcex colony1</td><td>0</td></tr> | ||
+ | <tr><td>11</td><td>〃2</td><td>0</td></tr> | ||
+ | <tr><td>12</td><td>〃3</td><td>0</td></tr> | ||
+ | <tr><td>13</td><td>〃4</td><td>0</td></tr> | ||
+ | <tr><td>14</td><td>9/6 PCR scfv+INPclos colony1</td><td>0</td></tr> | ||
+ | <tr><td>15</td><td>〃2</td><td>0</td></tr> | ||
+ | <tr><td>16</td><td>〃3</td><td>0</td></tr> | ||
+ | <tr><td>17</td><td>〃4</td><td>0</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a4/T--Kyoto--9_6_No81_Electrophoresis_Kosaka_09_30_11_22_50.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl</td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 0.2µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.1µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.02µl</td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/33/T--Kyoto--9_6_No82_General_Miyazaki_10_17_05_43_53.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">blotting CC<br>1'antibody time/buffer 1:3000 6ml 1h<br>2'antibody time/buffer 1:10000 10ml 1h<br>Blocking 1day<br>transfer 100mA 1h</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 10th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+</td><td>0.239</td><td>0.222</td><td></td></tr> | ||
+ | <tr><td>BclActl+</td><td>0.212</td><td>0.190</td><td></td></tr> | ||
+ | <tr><td>BclAcex-</td><td>0.239</td><td>0.233</td><td></td></tr> | ||
+ | <tr><td>BclActl-</td><td>0.221</td><td>0.210</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0906" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/6</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 11th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (NaCl)</td><td>0.202</td><td>0.176</td><td></td></tr> | ||
+ | <tr><td>BclAcex- (NaCl)</td><td>0.202</td><td>0.206</td><td></td></tr> | ||
+ | <tr><td>BclAcex- (NaCl)</td><td>0.202</td><td>0.264</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (PBS buffer)</td><td>0.212</td><td>0.179</td><td></td></tr> | ||
+ | <tr><td>BclAcex- (PBS buffer)</td><td>0.212</td><td>0.209</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (PBS buffer)</td><td>0.211</td><td>0.172</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>Bclascfv-BclaCBDcex colony5</td><td>vf12.5 vr12.5</td><td>10</td><td>KAPA2GFASTHSRM 12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃6</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃7</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃8</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃9</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃10</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>9/5 Bclascfv-BclaCBDclos 5</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃6</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>9/5 Bclascfv-INPCBDcex1</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃2</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃3</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃4</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>Bclascfv-INPCBDclos1</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃2</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃3</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃4</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c6/T--Kyoto--9_7_No83_Colony_PCR_%28typeA%29_Kosaka_09_30_11_33_11.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907” class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>Bcla-CBDcex</td><td>99.7</td><td>1.94</td><td>2.08</td></tr> | ||
+ | <tr><td>Bcla-CBDclos</td><td>73.5</td><td>1.96</td><td>2.15</td></tr> | ||
+ | <tr><td>INP-CBDcex</td><td>45.3</td><td>1.99</td><td>2.14</td></tr> | ||
+ | <tr><td>INP-CBDclos</td><td>49.6</td><td>1.89</td><td>1.89</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>8/5 miniprep INP-CBDcex</td><td>25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M/3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>8/5 miniprep INP-CBDclos</td><td>25</td><td>0</td><td>0</td><td>1</td><td>1</td><td>10×M/3</td><td>0</td><td>0</td><td>30</td></tr> | ||
+ | <tr><td>9/13 miniprep BclA-scFv1</td><td>22</td><td>0</td><td>1</td><td>0</td><td>1</td><td>10×H/3</td><td>3</td><td>0</td><td>30</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/5 Transformation BclascFv-BclaCBDcex colony5</td><td>LB(CP)/5ml</td></tr> | ||
+ | <tr><td>〃BclascFv-BclaCBDclos colony4</td><td>LB(CP)/5ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>SEM</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample</th><th>OD600</th><th>VLP(µl)</th></tr> | ||
+ | <tr><td>1</td><td>BclAscFv 3c5</td><td>0.990</td><td>1</td></tr> | ||
+ | <tr><td>2</td><td>BclAscFv 1c5</td><td>1.001</td><td>1</td></tr> | ||
+ | <tr><td>3</td><td>INPscFv 3c5</td><td>1.062</td><td>1</td></tr> | ||
+ | <tr><td>4</td><td>BclA CTL</td><td>1.053</td><td>1</td></tr> | ||
+ | <tr><td>5</td><td>INP CTL</td><td>0.952</td><td>1</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>Bcla-CBDcex</td><td>LB(CP)/30ml</td></tr> | ||
+ | <tr><td>Bcla-control</td><td> LB(CP)/30ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 12th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (PBS buffer)</td><td>0.195/0.192</td><td>0.188/0.189</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (PBS buffer)</td><td>0.198/0.195</td><td>0.192/0.189</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (CB buffer)</td><td>0.186/0.184</td><td>0.176/0.173</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (CB buffer)</td><td>0.172/0.169</td><td>0.151/0.155</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0907" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/7</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 13th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (PBS buffer) (Bemliese)</td><td>0.201/0.201</td><td>0.201/0.201</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (PBS buffer) (Bemliese)</td><td>0.224/0.223</td><td>0.218/0.215</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (CB buffer) (Bemliese)</td><td>0.266/0.257</td><td>0.243/0.242</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (CB buffer) (Bemliese)</td><td>0.178/0.177</td><td>0.157/0.149</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (PBS buffer) (Kimwipe)</td><td>0.203/0.201</td><td>0.200/0.196</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (PBS buffer) (Kimwipe)</td><td>0.221/0.220</td><td>0.224/0.229</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (CB buffer) (Kimwipe)</td><td>0.273/0.266</td><td>0.244/0.242</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (CB buffer) (Kimwipe)</td><td>0.172/0.172</td><td>0.154/0.154</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>9/7 restriction enzyme design BclA scFv X/Pcut</td><td>30</td></tr> | ||
+ | <tr><td>3</td><td>9/7 restriction enzyme design INP CBDcex S/Pcut</td><td>30</td></tr> | ||
+ | <tr><td>4</td><td>9/7 restriction enzyme design INP CBDclos S/Pcut</td><td>30</td></tr> | ||
+ | <tr><td>5</td><td>100bp ladder</td><td>5</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a8/T--Kyoto--9_8_No88_Gel_Extraction_Miyazaki_10_15_12_18_19.1.jpg"><img src="https://static.igem.org/mediawiki/2016/2/2a/T--Kyoto--9_8_No88_Gel_Extraction_Miyazaki_10_15_12_18_19.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/5 TransformationINPcex-scFv colony5</td><td>vf 1.25 vr1.25</td><td>10</td><td>KAPA2GFAST/12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃colony6</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃colony7</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃colony8</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>9/5 Transformation INPclos-BclascFv colony1</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃colony2</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃colony5</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃colony6</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/26/T--Kyoto--9_8_No89_Colony_PCR_%28typeA%29_Kosaka_10_02_01_52_26.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl</td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 0.2µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.1µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.02µl</td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/64/T--Kyoto--9_8_No90_General_Miyazaki_10_17_05_45_26.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">1'antibody time/buffer 1:3000 6ml 1h<br>2'antibody time/buffer 1:10000 10ml 1h<br>Blocking 1day<br>transfer 100mA 1h</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert/µl</th><th>Vector/µl</th><th>Ligation High/µl</th><th>Total</th></tr> | ||
+ | <tr><td>9/8 Gel Extraction BclA scFv X/Pcut /6</td><td>9/8 Gel Extraction INP CBDcex S/Pcut /1.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>9/8 Gel Extraction BclA scFv X/Pcut /6</td><td>9/8 Gel Extraction INP CBDcex S/Pcut /1.5</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>9/8 Ligation Bclascfv-INPCBDcex</td><td>2</td><td>DH5a/20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>〃BclascFv-INPCBDclos</td><td>2</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>9/7Miniprep BclaCBDcex+8/24 Miniprep J23105</td><td>1</td><td>〃</td><td>LB(CP+AMP)</td></tr> | ||
+ | <tr><td>〃Bcla-CBDclos+8/24 J23105</td><td>1</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>9/7 Miniprep INP-CBDcex+8/24 Miniprep J23105</td><td>1</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>9/7 Miniprep INP-CBDclos+8/24 Miniprep J23105</td><td>1</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>Bcla-CBDcex</td><td>LB(CP)/30ml</td></tr> | ||
+ | <tr><td>Bcla-CBDcontrol</td><td>LB(CP)/30ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0908" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/8</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 14th</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>CBDcex+ (CB buffer)</td><td>0.241/0.236</td><td>0.215/0.212</td><td></td></tr> | ||
+ | <tr><td>CBDctl+ (CB buffer)</td><td>0.182/0.180</td><td>0.159/0.159</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (CB buffer + Na2CO3)</td><td>0.235/0.235</td><td>0.177/0.177</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (CB buffer+Na2CO3)</td><td>0.227/0.218</td><td>0.155/0.156</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Glycerol Stock</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample /ml</th><th>glycerol /ml</th></tr> | ||
+ | <tr><td>BclA control /0.8</td><td>0.2</td></tr> | ||
+ | <tr><td>BclA control /0.8</td><td>0.2</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Glycerol Stock</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample /ml</th><th>glycerol /ml</th></tr> | ||
+ | <tr><td>INP control /0.8</td><td>0.2</td></tr> | ||
+ | <tr><td>INP control /0.8</td><td>0.2</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INPcex-scFv colony9</td><td>vf1.25 vr1.25</td><td>8.5</td><td>KAPA2GFAST 12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃colony10</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony11</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>9/5 transformation INPclos-scFv colony1</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃colony5</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/02/T--Kyoto--9_9_No96_Colony_PCR_%28typeA%29_Kosaka_10_02_02_29_44.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>scFv lc3</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl</td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 1µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.2µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.1µl</td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.02µl</td></tr> | ||
+ | <tr><td>8</td><td>Sano VLP 0.5µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>9</td><td>only SDS lysis buffer</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/7a/T--Kyoto--9_9_No98_General_Miyazaki_10_17_05_52_42.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">1'antibody time/buffer 1:3000 9ml 1h<br>2'antibody time/buffer 1:10000 10ml 1h<br>Blocking 1h<br>transfer 100mA 1h</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>scFv Bcla/1ul</td><td>271.8</td><td>2.09</td><td>2.15</td></tr> | ||
+ | <tr><td>VLP/1ul</td><td>1.67</td><td>1.98</td><td>- (A-280 10mmPath 1.670)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>BclaCBDcexJ23105</td><td>vf1.25 vr1.25</td><td>10</td><td>KAPA2GFAST HSRM withDye/12.5</td><td>25</td></tr> | ||
+ | <tr><td>9/6 Transformation BclaCBDclos J23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INPCBDcex J23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INPCBDcexJ23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/1b/T--Kyoto--9_9_No100_Colony_PCR_%28typeA%29_Kosaka_10_02_02_51_54.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/24 miniprep Bcla-his-cex colony2</td><td>0.1</td><td>DH5a/20ul</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Iguchi</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/5 transformation INPcex-BclascFv colony9</td><td>LB(CP)/5ml</td></tr> | ||
+ | <tr><td>9/5 transformation INPclos-BclascFv colony1</td><td> LB(CP)/5ml </td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0909" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/9</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/5 transformation Bcla CBDcex J23105 </td><td>vf1.0 vr1.0</td><td>23</td><td>Quick Taq/25</td><td>50</td></tr> | ||
+ | <tr><td>〃BclaCBDclos J23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INPCBDcex J23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃INPCBDclos J23105</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0912" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/9miniprep scFv Bcla lc3 </td><td>29ul</td><td></td><td>1</td><td>0</td><td>1</td><td>10×H/5</td><td>5</td><td>9</td><td>50</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0912" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/5 </td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>Bcla CBDcex j23105/15[1]</td><td>789 1140</td></tr> | ||
+ | <tr><td>3</td><td>〃[2]</td><td>〃</td></tr> | ||
+ | <tr><td>4</td><td>Bcla CBDclos J23105[1]</td><td>757 1140</td></tr> | ||
+ | <tr><td>5</td><td>〃[2]</td><td>〃</td></tr> | ||
+ | <tr><td>6</td><td>INP CBDcex J23105[1]</td><td>1664 1140</td></tr> | ||
+ | <tr><td>7</td><td>〃[2]</td><td>〃</td></tr> | ||
+ | <tr><td>8</td><td>INP CBDclos J23105[1]</td><td>1796 1140</td></tr> | ||
+ | <tr><td>9</td><td>〃[2]</td><td>〃</td></tr> | ||
+ | <tr><td>10</td><td>100bp ladder</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4f/T--Kyoto--9_12_No105_Electrophoresis_Kosaka_10_02_03_10_40.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0912" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>Bcla CBDcex J23105[1]</td><td>vf0.5 vr0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>〃[2]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃[3]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃[4]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>Bcla CBDclos J23105[1]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃[2]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃[3]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | <tr><td>〃[4]</td><td>〃</td><td>〃</td><td>〃</td><td>〃</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Kosaka --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0912" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INP CBDcex J23105</td><td>vf0.5 vr0.5</td><td>11.5</td><td>Quick Taq12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP〃[2]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃[3]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃[4]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃clos[1]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃[2]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃[3]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | <tr><td>〃[4]</td><td>=</td><td>=</td><td>=</td><td>=</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0913" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 1µl</td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 1µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.2µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.1µl</td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.05µl</td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.02µl</td></tr> | ||
+ | <tr><td>8</td><td>Sano VLP 0.5µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>9</td><td>only SDS lysis buffer</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c8/T--Kyoto--9_13_No112_General_Miyazaki_10_17_05_58_13.1.jpg"><img src="https://static.igem.org/mediawiki/2016/3/3c/T--Kyoto--9_13_No112_General_Miyazaki_10_17_05_58_13.2.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">1'antibody time/buffer 1:3000 9ml 1h<br>2'antibody time/buffer 1:10000 10ml 1h<br>Blocking 1h<br>transfer 100mA 1h<br>30ml TBS-T/blocking reagent 1.5g</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/9 transformation BclA-CBDcex</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/24 transformation BclA-CBDclos</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/24 transformation INP-CBDcex</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/20 transformation </td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0914" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl+INP-scFv 100µl </td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 0.5µl+BAN-scFv 100µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.2µl +BAN-scFv 100µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl </td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl </td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.2µl +BANcontrol100µl</td></tr> | ||
+ | <tr><td>8</td><td>Sano VLP 0.2µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>9</td><td>Sano VLP 0.5µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>10</td><td>Marker</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6a/T--Kyoto--9_14_No114_General_Miyazaki_10_17_06_06_07.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight<br>2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h<br>Blocking 1h<br>transfer 100mA 1h<br>25ml TBS-T/blocking reagent 1.3g</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0915" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>1</td><td>Marker</td></tr> | ||
+ | <tr><td>2</td><td>Sano VLP 0.5µl+INP-scFv 100µl </td></tr> | ||
+ | <tr><td>3</td><td>Sano VLP 0.5µl+BAN-scFv 100µl</td></tr> | ||
+ | <tr><td>4</td><td>Sano VLP 0.2µl +BAN-scFv 100µl</td></tr> | ||
+ | <tr><td>5</td><td>Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl </td></tr> | ||
+ | <tr><td>6</td><td>Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl </td></tr> | ||
+ | <tr><td>7</td><td>Sano VLP 0.2µl +BANcontrol100µl</td></tr> | ||
+ | <tr><td>8</td><td>Sano VLP 0.2µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>9</td><td>Sano VLP 0.5µl 95℃ without boiling</td></tr> | ||
+ | <tr><td>10</td><td>Marker</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/8f/T--Kyoto--9_15_No116_General_Miyazaki_10_17_06_11_34.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight<br>2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h<br>Blocking 1h<br>transfer 100mA 1h<br>25ml TBS-T/blocking reagent 1.3g</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0915" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/7 BclA scFv 3C5 9/7 2 </td><td>LB(CP) /20mL</td></tr> | ||
+ | <tr><td>9/14 BclA cex (9/9 transformation)</td><td>LB(CP) /20mL</td></tr> | ||
+ | <tr><td>9/7 INP scFv 3C5</td><td>LB(CP) /20mL</td></tr> | ||
+ | <tr><td>9/9 BclA scFv 1c3</td><td>LB(CP) /20mL</td></tr> | ||
+ | <tr><td>9/7 INP CTL</td><td>LB(CP) /20mL</td></tr> | ||
+ | <tr><td>9/7 BclA CTL 2</td><td>LB(CP) /20mL</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0915" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA CBDclos glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDcex glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-CBDclos glycerol stock</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0916" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>8/20 J23105 RFP 1/100 </td><td>20</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0919" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Takimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/24 BAN-cex</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/5 INP-cex</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/8 INP HIsR</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/19 BAN-clos</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/5 INP-clos</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>2/4 E1010</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>8/18 BclA-scFv</td><td>1</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0919" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/19</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Takimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/15 Liquid culture BclA scFv 3C5[2]</td><td>LB(CP) /20</td></tr> | ||
+ | <tr><td>9/19 Liquid culture Bcla CTL</td><td>LB(CP) /20</td></tr> | ||
+ | <tr><td>9/15 Liquid culture INP-scFv 3C5</td><td>LB(CP) /20</td></tr> | ||
+ | <tr><td>9/15 Liquid culture INP CTL</td><td>LB(CP) /20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0920" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/19 liquid culture BclA-scFv 3C5-2</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 liquid culture BclA-ctl</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 liquid culture INP-scFv 3C5</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 liquid culture INP-ctl</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0920" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker</td></tr> | ||
+ | <tr><td>1</td><td>INPNC-6His-scFv 300 µl+VLP3 µl</td></tr> | ||
+ | <tr><td>2</td><td>INPNC-6His-scFv 300 µl+PBS3 µl </td></tr> | ||
+ | <tr><td>3</td><td>INPNC-6His-ctl300 µl+VLP3 µl </td></tr> | ||
+ | <tr><td>4</td><td>BclA-6His-scFv 300 µl+VLP3 µl </td></tr> | ||
+ | <tr><td>5</td><td>BclA-6His-scFv 300 µl+PBS3 µl </td></tr> | ||
+ | <tr><td>6</td><td>BclA-6His-ctl 300 µl+VLP3 µl </td></tr> | ||
+ | <tr><td>7</td><td>VLP0.5 µl</td></tr> | ||
+ | <tr><td>8</td><td>VLP0.2 µl</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--9_20_No124_General_Miyazaki_10_17_06_17_54.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">Transfer ;60 min 100 mA(CC)<br>Blocking ;1h<br>1’antibody ;anti-NoV pAb(mouse)<br>1:1000 with blocking buffer O/N<br>2’antibody ;anti-mouse HRP <br>1:2500 with blocking buffer 1 h<br>Detection time 80 sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0920" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/20</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>BclA-clos glycerol stock</td><td>-</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP-clos glycerol stock</td><td>-</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>8℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 miniprep INP-His9 /0.2</td><td>VF /0.75</td><td>m.scFv Rv /0.75</td><td>-</td><td>10.8</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His9 /0.2</td><td>m.scFv Fw /0.75</td><td>VR /0.75</td><td>-</td><td>10.8</td><td>KAPA HiFi Ready Mix /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">VF 10µM VR 10µM</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>100bp ladder </td><td>5</td></tr> | ||
+ | <tr><td>3</td><td>9/21 PCR INP-His9-1</td><td>25</td></tr> | ||
+ | <tr><td>4</td><td>9/21 PCR INP-His9-2</td><td>25</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/53/T--Kyoto--9_21_No129_Gel_Extraction_Notsu_10_03_01_11_36.1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b2/T--Kyoto--9_21_No129_Gel_Extraction_Notsu_10_03_01_11_36.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN-scFv 3C5[2]</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN-CTL </td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP-scFv 3C5</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP-CTL</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/15 BAN- scFv 1C3</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>9/15 BAN-CBDcex</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker</td></tr> | ||
+ | <tr><td>1</td><td>INPNC-6His-scFv 200 µl+VLP2 µl</td></tr> | ||
+ | <tr><td>2</td><td>INPNC-6His-scFv 200 µl+PBS2µl(NC)</td></tr> | ||
+ | <tr><td>3</td><td>INPNC-6His-ctl200 µl+VLP2 µl(NC)</td></tr> | ||
+ | <tr><td>4</td><td>BclA-6His-scFv200 ul(Vector pSB3C5)+VLP2 ul</td></tr> | ||
+ | <tr><td>5</td><td>BclA-6His-scFv 200 µl+VLP2 µl (NC)</td></tr> | ||
+ | <tr><td>6</td><td> BclA-6His-scFv(Vector pSB1C3)+VLP2 ul</td></tr> | ||
+ | <tr><td>7</td><td> BclA-6His-CBDcex 200 ul + VLP2 ul (NC)</td></tr> | ||
+ | <tr><td>8</td><td>PBS200 ul +VLP2 ul (NC)</td></tr> | ||
+ | <tr><td>9</td><td>VLP1 ul (PC) </td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/16/T--Kyoto--9_21_No131_General_Miyazaki_10_17_06_47_39.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">Transfer ;60 min 100 mA(CC)<br>Blocking ;1h<br>1’antibody ;anti-NoV pAb(mouse)<br>1:1000 with blocking buffer O/N<br>2’antibody ;anti-mouse HRP <br>1:2500 with blocking buffer 1 h<br>Detection time 60 sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction(Measurement)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample/(µl)</th><th>Concentration(µg/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/21 PCR INP-His9-1</td><td>19.3</td><td>1.63</td><td>0.54</td></tr> | ||
+ | <tr><td>9/21 PCR INP-His9-2</td><td>48.6</td><td>1.82</td><td>0.24</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Steps)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Steps</th><th>Temparature</th><th>Time</th><th>Cycle</th></tr> | ||
+ | <tr><td>Predenature</td><td>95℃</td><td>5min</td><td>1</td></tr> | ||
+ | <tr><td>Denature</td><td>98℃</td><td>20sec</td><td>30</td></tr> | ||
+ | <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>30</td></tr> | ||
+ | <tr><td>Extension</td><td>72℃</td><td>30sec</td><td>30</td></tr> | ||
+ | <tr><td>Final Elongation</td><td>72℃</td><td>3min</td><td>1</td></tr> | ||
+ | <tr><td>Final Holding</td><td>8℃</td><td>-</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0921" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/21</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/21 Gel Extract INP-His9-1 /1µl+9/21 Gel Extract INP-His9-2 /0.5µl</td><td>VF /0.75 </td><td>VR /0.75</td><td>-</td><td>9.5</td><td>KAPA HiFi /12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0922" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/22</h3></span> | ||
+ | <span class="keln_exp"><h4>growth carve</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>0h</th><th>0.5h</th><th>1h</th><th>1.5h</th><th>2h</th><th>3h</th><th>4h</th><th>5h</th><th>6h</th><th>7h</th><th>8h</th></tr> | ||
+ | <tr><td>BAM scFv [1]</td><td>-0.048</td><td>-0.047</td><td>-0.048</td><td>1.245</td><td>-0.317</td><td>-0.308</td><td>-0.149</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAM scFv [2]</td><td>-0.036</td><td>1.512</td><td>-0.830</td><td>-0.309</td><td>-0.314</td><td>-0.127</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP scFv [1]</td><td>-0.047</td><td>-0.050</td><td>-0.644</td><td>1.045</td><td>-0.307</td><td>-0.153</td><td>-0.142</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP scFv [2]</td><td>-0.038</td><td>-0.o25</td><td>-0.186</td><td>-0.280</td><td>0.000</td><td>-0.289</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CBDcex [1]</td><td>-0.045</td><td>-0.048</td><td>-0.044</td><td>1.089</td><td>-0.322</td><td>-0.284</td><td>-0.296</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CBDcex [2]</td><td>-0.034</td><td>1.204</td><td>-1.057</td><td>-0.311</td><td>0.268</td><td>-0.285</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CBDclos [1]</td><td>0.035</td><td>-0.037</td><td>-0.035</td><td>1.261</td><td>-0.323</td><td>-0.160</td><td>-0.287</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CBDclos [2]</td><td>-0.018</td><td>1.306</td><td>-0.050</td><td>-0.282</td><td>-0.128</td><td>-0.293</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CBDcex [1]</td><td>-0.020</td><td>-0.027</td><td>-0.016</td><td>1.245</td><td>-0.295</td><td>-0.263</td><td>-0.288</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CBDcex [2]</td><td>-0.027</td><td>1.608</td><td>0.016</td><td>-0.287</td><td>-0.224</td><td>-0.294</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CBDclos [1]</td><td>-0.034</td><td>-0.034</td><td>-0.032</td><td>1.026</td><td>-0.318</td><td>-0.235</td><td>-0.270</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CBDclos [2]</td><td>-0.026</td><td>1.415</td><td>0.012</td><td>-0.297</td><td>-0.047</td><td>-0.272</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CTL [1]</td><td>-0.039</td><td>-0.039</td><td>-0.036</td><td>1.274</td><td>-0.314</td><td>-0.230</td><td>-0.271</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>BAN CTL [2]</td><td>-0.056</td><td>1.530</td><td>-0.060</td><td>-0.284</td><td>-0.084</td><td>-3.11</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CTL [1]</td><td>-0.042</td><td>-0.043</td><td>-0.042</td><td>1.134</td><td>-0.235</td><td>-0.181</td><td>-0.296</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>INP CTL [2]</td><td>-0.042</td><td>-1.488</td><td>-0.030</td><td>-0.288</td><td>-0.109</td><td>-0.295</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>E1010 [1]</td><td>-0.041</td><td>-0.040</td><td>-0.036</td><td>1.171</td><td>-0.322</td><td>-0.252</td><td>-0.271</td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>E1010 [2]</td><td>-0.030</td><td>1.563</td><td>-0.061</td><td>-0.267</td><td>0.286</td><td>-0.267</td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | <tr><td>blank [1]/[2]</td><td></td><td></td><td></td><td> /-0.307</td><td>-0.306/-0.305</td><td>-0.307/-0.271</td><td>0.306/</td><td></td><td></td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Notsu --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0922" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/22</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>9/21 liquid culture BAN-CBDcex</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture INP-ctl</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture INP-CBDcex</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture INP-CBDclos</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture BAN-scFv 3C5</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture INP-scFv 3C5</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/19 liquid culture BAN-CBDclos</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/15 liquid culture BAN-ctl</td><td>LB(AMP)</td></tr> | ||
+ | <tr><td>9/16 transformation J23105-RFP </td><td>LB(AMP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>SEM</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample(µl)</th><th>VLP(µl)</th><th>PBS</th><th>centrifugal force</th></tr> | ||
+ | <tr><td>1</td><td>INP scFv /25</td><td>2.5</td><td>0</td><td>×3000g</td></tr> | ||
+ | <tr><td>2</td><td>INP CTL /25</td><td>2.5</td><td>0</td><td>×3000g</td></tr> | ||
+ | <tr><td>3</td><td>BclA scFv /25</td><td>2.5</td><td>0</td><td>×3000g</td></tr> | ||
+ | <tr><td>4</td><td>BclA CTL /25</td><td>2.5</td><td>0</td><td>×3000g</td></tr> | ||
+ | <tr><td>5</td><td>BclA scFv /25</td><td>0</td><td>2.5</td><td>×3000g</td></tr> | ||
+ | <tr><td>6</td><td>INP scFv /25</td><td>2.5</td><td>0</td><td>×200g</td></tr> | ||
+ | <tr><td>7</td><td>INP CTL /25</td><td>2.5</td><td>0</td><td>×200g</td></tr> | ||
+ | <tr><td>8</td><td>BclA scFv /25</td><td>2.5</td><td>0</td><td>×200g</td></tr> | ||
+ | <tr><td>9</td><td>BclA CTL /25</td><td>2.5</td><td>0</td><td>×200g</td></tr> | ||
+ | <tr><td>10</td><td>BclA scFv /25</td><td>0</td><td>2.5</td><td>×200g</td></tr> | ||
+ | <tr><td>11</td><td>0</td><td>2.5</td><td>0</td><td>×200g</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>RNA extraction</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample name</th></tr> | ||
+ | <tr><td>1</td><td>BclA-CBD cex</td></tr> | ||
+ | <tr><td>2</td><td>BclA-CBDclos</td></tr> | ||
+ | <tr><td>3</td><td>INP-CBDcex</td></tr> | ||
+ | <tr><td>4</td><td>INP-CBDclos</td></tr> | ||
+ | <tr><td>5</td><td>BclA-scFv</td></tr> | ||
+ | <tr><td>6</td><td>INP-scFv</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>growth carve</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>0h</th><th>0.5h</th><th>1h</th><th>1.5h</th><th>2h</th><th>3h</th><th>4h</th><th>5h</th><th>6h</th><th>7h</th><th>8h</th><th>9h</th><th>10h</th></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN scFv[1]</td><td>0.003</td><td>0.001</td><td>0.000</td><td>0.008</td><td>0.015</td><td>0.009</td><td>0.013</td><td>0.016</td><td>0.015</td><td>0.022</td><td>0.062</td><td>0.020</td><td>0.027</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN scFv[2]</td><td>0.013</td><td>0.012</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.001</td><td>0.011</td><td>0.011</td><td>0.015</td><td>0.022</td><td>0.027</td><td>0.057</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP scFv[1]</td><td>0.012</td><td>0.000</td><td>0.000</td><td>0.011</td><td>0.006</td><td>0.006</td><td>0.007</td><td>0.008</td><td>0.013</td><td>0.025</td><td>0.036</td><td>0.098</td><td>0.076</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP scFv[2]</td><td>0.007</td><td>0.005</td><td>0.000</td><td>0.002</td><td>0.003</td><td>0.002</td><td>0.012</td><td>0.012</td><td>0.019</td><td>0.029</td><td>0.034</td><td>0.047</td><td>0.072</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN cex[1]</td><td>0.014</td><td>0.005</td><td>0.010</td><td>0.008</td><td>0.011</td><td>0.009</td><td>0.014</td><td>0.014</td><td>0.016</td><td>0.029</td><td>0.038</td><td>0.043</td><td>0.083</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN cex[2]</td><td>0.011</td><td>0.011</td><td>0.010</td><td>0.008</td><td>0.004</td><td>0.004</td><td>0.020</td><td>0.021</td><td>0.036</td><td>0.047</td><td>0.065</td><td>0.087</td><td>0.109</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN clos[1]</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.002</td><td>0.009</td><td>0.003</td><td>0.009</td><td>0.014</td><td>0.019</td><td>0.014</td><td>0.016</td><td>0.024</td><td>0.046</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN clos[2]</td><td>0.005</td><td>0.007</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.003</td><td>0.012</td><td>0.022</td><td>0.020</td><td>0.035</td><td>0.055</td><td>0.086</td><td>0.117</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP cex[1]</td><td>0.187</td><td>0.000</td><td>0.001</td><td>0.007</td><td>0.011</td><td>0.006</td><td>0.013</td><td>0.016</td><td>0.020</td><td>0.023</td><td>0.037</td><td>0.048</td><td>0.068</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP cex[2]</td><td>0.002</td><td>0.009</td><td>0.000</td><td>0.001</td><td>0.001</td><td>0.002</td><td>0.015</td><td>0.017</td><td>0.023</td><td>0.036</td><td>0.053</td><td>0.080</td><td>0.111</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP clos[1]</td><td>0.006</td><td>0.000</td><td>0.000</td><td>0.023</td><td>0.011</td><td>0.003</td><td>0.011</td><td>0.013</td><td>0.016</td><td>0.022</td><td>0.033</td><td>0.058</td><td>0.068</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP clos[2]</td><td>0.029</td><td>0.028</td><td>0.000</td><td>0.009</td><td>0.000</td><td>0.016</td><td>0.001</td><td>0.007</td><td>0.008</td><td>0.014</td><td>0.020</td><td>0.029</td><td>0.074</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN CTL[1]</td><td>0.004</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.002</td><td>0.000</td><td>0.002</td><td>0.012</td><td>0.015</td><td>0.012</td><td>0.027</td><td>0.055</td><td>0.081</td></tr> | ||
+ | <tr><td>9/19 Liquid culture BAN CTL[2]</td><td>0.002</td><td>0.002</td><td>0.001</td><td>0.000</td><td>0.001</td><td>0.005</td><td>0.017</td><td>0.024</td><td>0.031</td><td>0.042</td><td>0.059</td><td>0.073</td><td>0.113</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP CTL[1]</td><td>0.011</td><td>0.000</td><td>0.032</td><td>0.013</td><td>0.017</td><td>0.010</td><td>0.015</td><td>0.033</td><td>0.031</td><td>0.035</td><td>0.037</td><td>0.019</td><td>0.026</td></tr> | ||
+ | <tr><td>9/19 Liquid culture INP CTL[2]</td><td>0.013</td><td>0.020</td><td>0.000</td><td>0.001</td><td>0.000</td><td>0.003</td><td>0.009</td><td>0.016</td><td>0.021</td><td>0.026</td><td>0.044</td><td>0.059</td><td>0.136</td></tr> | ||
+ | <tr><td>9/19 Liquid culture E1010[1]</td><td>0.015</td><td>0.016</td><td>0.014</td><td>0.011</td><td>0.016</td><td>0.018</td><td>0.032</td><td>0.066</td><td>0.108</td><td>0.173</td><td>0.258</td><td>0.361</td><td>0.393</td></tr> | ||
+ | <tr><td>9/19 Liquid culture E1010[2]</td><td>0.064</td><td>0.010</td><td>0.007</td><td>0.011</td><td>0.012</td><td>0.019</td><td>0.029</td><td>0.041</td><td>0.061</td><td>0.097</td><td>0.150</td><td>0.180</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/21 OEPCR/25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6f/T--Kyoto--9_23_No139_Electrophoresis_Miyazaki_10_06_03_28_24.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>OEPCR</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample1 /µl</th><th>sample2 /µl</th><th>primer Fw /µl</th><th>primer Rv /µl</th><th>milliQ /µl</th><th>KAPA HiFi /µl</th><th>total</th></tr> | ||
+ | <tr><td>9/21 Gel Extract1 /1</td><td>9/21 Gel Extract2 /0.5</td><td>VF/ 0.75</td><td>VR /0.75</td><td>9.5</td><td>12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/3</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/23 OEPCR1 /25</td><td></td></tr> | ||
+ | <tr><td>3</td><td>9/23 OEPCR2 /25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/82/T--Kyoto--9_23_No141_Electrophoresis_Miyazaki_10_06_03_34_18.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0923" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/23</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv9 /0.4</td><td>m.scFv Fw/1.25</td><td>VR/1.25</td><td>KAPA2GFAST/12.5</td><td>9.6</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv9 /0.4</td><td>m.scFv Fw/1.25</td><td>VR/1.25</td><td>KAPA2GFAST/12.5</td><td>9.6</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv9 /0.4</td><td>VF/1.25</td><td>m.scFv Rv/1.25</td><td>KAPA2GFAST/12.5</td><td>9.6</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv9 /0.4</td><td>VF/1.25</td><td>m.scFv Rv/1.25</td><td>KAPA2GFAST/12.5</td><td>9.6</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>9/23 Gel Extraction INP-scFv m.PCR/1.5</td><td>VF/1.25</td><td>VR/1.25</td><td>KAPA2GFAST/12.5</td><td>8.5</td><td>0</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/4</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/23PCR1 /25</td><td></td></tr> | ||
+ | <tr><td>3</td><td>9/23PCR2 /25</td><td></td></tr> | ||
+ | <tr><td>4</td><td>9/23PCR3 /25</td><td></td></tr> | ||
+ | <tr><td>5</td><td>9/23PCR4 /25</td><td></td></tr> | ||
+ | <tr><td>6</td><td>9/23PCR5 /25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/68/T--Kyoto--9_26_No143_Electrophoresis_Miyazaki_10_06_03_46_00.1.jpg"><img src="https://static.igem.org/mediawiki/2016/1/18/T--Kyoto--9_26_No143_Electrophoresis_Miyazaki_10_06_03_46_00.2.jpg"> | ||
+ | </div> | ||
+ | |||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>9/26 electrophoresis lane 4,5</td><td></td><td>0.357g</td></tr> | ||
+ | <tr><td>9/26 electrophoresis lane 2,3</td><td></td><td>0.354g</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | |||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/26 Gel Extract lane4,5 /2</td><td>VF/0.75</td><td>VR/0.75</td><td>2×KAPA HiFi Hotstart ReadyMix/12.5</td><td>7</td><td></td><td>25</td></tr> | ||
+ | <tr><td>9/26 Gel Extract lane2,3 /2</td><td>VF/0.75</td><td>VR/0.75</td><td>2×KAPA HiFi Hotstart ReadyMix/12.5</td><td>7</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>INP-scFv/30ml</td><td>LB</td></tr> | ||
+ | <tr><td>INP-CTL/30ml</td><td>LB</td></tr> | ||
+ | <tr><td>BAN-scFv/30ml</td><td>LB</td></tr> | ||
+ | <tr><td>Ban CTL/30ml</td><td>LB</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>Bcla-cex 8/5 miniprep</td><td></td><td>BL21</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>Bcla-CTL 8/4 miniprep</td><td></td><td>BL21</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>Bcla-cex 8/5 miniprep</td><td></td><td>BL21</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>Bcla-CTL 8/4 miniprep</td><td></td><td>BL21</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0926" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/26</h3></span> | ||
+ | <span class="keln_exp"><h4>RNA extraction</h4></span> | ||
+ | <span class="keln_researcher">Isoda</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample</th><th>conc.µg/µl</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>4</td><td>BclAcex</td><td>74.73</td><td>1.94</td><td>1.61</td></tr> | ||
+ | <tr><td>2</td><td>INPcex</td><td>83</td><td>1.97</td><td>1.38</td></tr> | ||
+ | <tr><td>5</td><td>INPclos</td><td>120.21</td><td>1.99</td><td>1.94</td></tr> | ||
+ | <tr><td>3</td><td>INPclos</td><td>58.23</td><td>1.98</td><td>1.84</td></tr> | ||
+ | <tr><td>6</td><td>BclAscFV</td><td>74.76</td><td>1.96</td><td>1.84</td></tr> | ||
+ | <tr><td>7</td><td>INPscFv</td><td>80.6</td><td>1.97</td><td>1.97</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BclA-cex /250ml</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>BclA-CTL /250ml</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>growth carve</h4></span> | ||
+ | <span class="keln_researcher">Sassa, Takimoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>0h</th><th>0.5h</th><th>1h</th><th>1.5h</th><th>2h</th><th>3h</th><th>4h</th><th>5h</th><th>6h</th><th>7h</th><th>8h</th></tr> | ||
+ | <tr><td>9/21Liquid culture BAN scFv</td><td>0.113</td><td>0.170</td><td>0.266</td><td>0.408</td><td>0.599</td><td>0.893</td><td>0.164(1.64)</td><td>0.169(1.69)</td><td>0.232(2.32)</td><td>0.234(2.34)</td><td>0.221(2.21)</td></tr> | ||
+ | <tr><td>9/22Liquid culture BAN cex</td><td>0.109</td><td>0.147</td><td>0.219</td><td>0.374</td><td>0.515</td><td>0.873</td><td>0.146(1.46)</td><td>0.160(1.60)</td><td>0.176(1.76)</td><td>0.231(2.31)</td><td>0.260(2.60)</td></tr> | ||
+ | <tr><td>9/19Liquid culture BAN clos</td><td>0.097</td><td>0.109</td><td>0.184</td><td>0.338</td><td>0.486</td><td>0.744</td><td>0.130(1.30)</td><td>0.173(1.73)</td><td>0.199(1.99)</td><td>0.203(2.03)</td><td>0.274(2.74)</td></tr> | ||
+ | <tr><td>9/21Liquid culture BAN CTL</td><td>0.106</td><td>0.170</td><td>0.252</td><td>0.304</td><td>0.553</td><td>0.797</td><td>0.177(1.77)</td><td>0.155(1.55)</td><td>0.216(2.16)</td><td>0.236(2.36)</td><td>0.257(2.57)</td></tr> | ||
+ | <tr><td>9/21Liquid culture INP scFv</td><td>0.088</td><td>0.083</td><td>0.101</td><td>0.122</td><td>0.299</td><td>0.409</td><td>0.073(0.73)</td><td>0.121(1.21)</td><td>0.140(1.40)</td><td>0.143(1.43)</td><td>0.195(1.95)</td></tr> | ||
+ | <tr><td>9/15Liquid culture INP cex</td><td>0.102</td><td>0.116</td><td>0.145</td><td>0.207</td><td>0.320</td><td>0.626</td><td>0.170(1.70)</td><td>0.143(1.43)</td><td>0.176(1.76)</td><td>0.188(1.88)</td><td>0.238(2.38)</td></tr> | ||
+ | <tr><td>9/22Liquid culture INP clos</td><td>---</td><td>0.133</td><td>0.209</td><td>0.373</td><td>0.572</td><td>0.930</td><td>0.156(1.56)</td><td>0.201(2.01)</td><td>0.246(2.46)</td><td>0.237(2.37)</td><td>0.293(2.93)</td></tr> | ||
+ | <tr><td>9/21Liquid culture INP CTL</td><td>0.112</td><td>0.155</td><td>0.237</td><td>0.409</td><td>0.582</td><td>0.955</td><td>0.153(1.53)</td><td>0.209(2.09)</td><td>0.268(2.68)</td><td>0.288(2.88)</td><td>0.276(2.76)</td></tr> | ||
+ | <tr><td>9/21Liquid culture E1010</td><td>0.073</td><td>0.091</td><td>0.149</td><td>0.265</td><td>0.396</td><td>0.706</td><td>0.157(1.57)</td><td>0.182(1.82)</td><td>0.222(2.22)</td><td>0.222(2.22)</td><td>0.246(2.46)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">rpm 160<br>total10mL<br>CP 2mL/1L<br>measured 10 fold dilution after 4h </p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker(Wako)</td></tr> | ||
+ | <tr><td>1</td><td>BclA6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) </td></tr> | ||
+ | <tr><td>2</td><td>BclA6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) </td></tr> | ||
+ | <tr><td>3</td><td>INPNC6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) </td></tr> | ||
+ | <tr><td>4</td><td>INPNC6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) </td></tr> | ||
+ | <tr><td>5</td><td>BclA6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) </td></tr> | ||
+ | <tr><td>6</td><td>BclA6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) </td></tr> | ||
+ | <tr><td>7</td><td>INPNC6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) </td></tr> | ||
+ | <tr><td>8</td><td>INPNC6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) </td></tr> | ||
+ | <tr><td>9</td><td>PBS 300µl+supernatant of NoVLP 3 µl (NC) (washed by PBS)</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/3d/T--Kyoto--9_27_No149_General_Miyazaki_10_17_06_53_43.png"> | ||
+ | </div> | ||
+ | <p class="keln_note">Transfer ;60 min 100 mA(CC)<br>Blocking ;1h<br>1’antibody ;anti-NoV pAb(mouse)<br>1:1000 with blocking buffer O/N<br>2’antibody ;anti-mouse HRP <br>1:2500 with blocking buffer 1 h<br>Detection time 60 sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara, Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker(Wako) 5µl</td></tr> | ||
+ | <tr><td>1</td><td>BBa_E1010(NC) </td></tr> | ||
+ | <tr><td>2</td><td>BclA-6His-CBDclos (17 kDa) </td></tr> | ||
+ | <tr><td>3</td><td>INPNC-6His-CBDclos(47 kDa) </td></tr> | ||
+ | <tr><td>4</td><td>BclA-6His-CBDcex (17 kDa) </td></tr> | ||
+ | <tr><td>5</td><td>INPNC-6His-CBDcex (47 kDa) </td></tr> | ||
+ | <tr><td>6</td><td>BclA-6His-scFv (33 kDa) </td></tr> | ||
+ | <tr><td>7</td><td>INPNC-6His-scFv (63 kDa) </td></tr> | ||
+ | <tr><td>8</td><td>BBa_K875004 (PC) Lpp-OmpA-scFv-6His (43kDa) </td></tr> | ||
+ | <tr><td>Marker</td><td> M.Marker(Wako) 3µl</td></tr> | ||
+ | <tr><td>9</td><td>BBa_K875004 (PC) Lpp-OmpA-scFv-6His(43 kDa) </td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/2a/T--Kyoto--9_27_No149_General_Miyazaki_10_17_07_01_58.png"> | ||
+ | </div> | ||
+ | <p class="keln_note">Used membrane fraction from 100ml culture at logarithmic growth phase.<br><br>Transfer ;60min 100mA(CC)<br>Blocking ;1h<br>1’antibody ;anti6His(mouse)<br>1:1000 with blocking buffer O/N<br>2’antibody ;anti-mouse HRP <br>1:2500 with TBST 1h<br>Detection time 380sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Reverse Transcription</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>Template/µl</th><th>gDNA wipeout buffer/µl</th><th>RNAase free water/µl</th><th>total/µl</th></tr> | ||
+ | <tr><td>A1</td><td>BclAcex/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>A2</td><td>BclAclos/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>A3</td><td>INPcex/8.32</td><td>2</td><td>3.68</td><td>14</td></tr> | ||
+ | <tr><td>A4</td><td>INPclos/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>A5</td><td>BclAscFv/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>A6</td><td>INPscFv/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>B1</td><td>BclAcex/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>B2</td><td>BclAclos/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>B3</td><td>INPcex/8.32</td><td>2</td><td>3.68</td><td>14</td></tr> | ||
+ | <tr><td>B4</td><td>INPclos/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>B5</td><td>BclAscFv/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | <tr><td>B6</td><td>INPscFv/12</td><td>2</td><td>0</td><td>14</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Reverse Transcription</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>template/µl</th><th>RTase/µl</th><th>RNAase free water/µl</th><th>RT buffer/µl</th><th>Primer Mix/µl</th><th>Total</th></tr> | ||
+ | <tr><td>A1</td><td>BclAcex/14</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>A2</td><td>BclAclos/14</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>A3</td><td>INPcex/14</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>A4</td><td>INPclos/14</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>A5</td><td>BclAscFv/14</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>A6</td><td>INPscFv/12</td><td>1</td><td>0</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B1</td><td>BclAcex/14</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B2</td><td>BclAclos/14</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B3</td><td>INPcex/14</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B4</td><td>INPclos/14</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B5</td><td>BclAscFv/14</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | <tr><td>B6</td><td>INPscFv/12</td><td>0</td><td>1</td><td>4</td><td>1</td><td>20</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/26 PCR INP- His scFv/25</td><td></td></tr> | ||
+ | <tr><td>3</td><td>9/26 PCR INP- His scFv/25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/ed/T--Kyoto--9_27_No152_Electrophoresis_Miyazaki_10_06_04_10_12.1.jpg"><img src="https://static.igem.org/mediawiki/2016/a/a0/T--Kyoto--9_27_No152_Electrophoresis_Miyazaki_10_06_04_10_12.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>-</td><td>9/27 Electrophoresis INP-His-scFv</td><td>0.7660g</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">nanodrop<br>17.8ng/nl<br>260/280 1.79<br>260/230 0.31</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0927" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/27</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 15th</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>BclAcex+ (CB buffer)</td><td>0.181/0.212/0.204</td><td>0.254/0.255/0.254</td><td>0/0/0</td></tr> | ||
+ | <tr><td>BclAcex- (CB buffer)</td><td>0.208/0.275/0.228</td><td>0.160/0.171/0.162</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (CB buffer)</td><td>0.178/0.176/0.171</td><td>0.082/0.085/0.082</td><td>0.022/0.02/0.02</td></tr> | ||
+ | <tr><td>BclActl- (CB buffer)</td><td>0.218/0.214/0.216</td><td>0.161/0.160/0.159</td><td></td></tr> | ||
+ | <tr><td>BclAcex+ (PBS buffer)</td><td>0.212/0.211/0.210</td><td>0.333/0.334/0.333</td><td>0.022/0.022/0.021</td></tr> | ||
+ | <tr><td>BclAcex- (PBS buffer)</td><td>0.203/0.202/0.201</td><td>0.178/0.178/0.180</td><td></td></tr> | ||
+ | <tr><td>BclActl+ (PBS buffer)</td><td>0.182/0.181/0.180</td><td>0.253/0.246/0.246</td><td>0.021/0.021/0.021</td></tr> | ||
+ | <tr><td>BclActl- (PBS buffer)</td><td>0.208/0.205/0.208</td><td>0.192/0.194/0.194</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0929" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/29</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>9/27 Gel Extract/2</td><td>VF/0.75</td><td>VR/0.75</td><td>2×KAPA HiFi Hotstart ReadyMix/12.5</td><td>9</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0929" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>BAN scFv</td><td>LB</td></tr> | ||
+ | <tr><td>INP clos</td><td>LB</td></tr> | ||
+ | <tr><td>BAN clos</td><td>LB</td></tr> | ||
+ | <tr><td>INP cex</td><td>LB</td></tr> | ||
+ | <tr><td>BAN cex</td><td>LB</td></tr> | ||
+ | <tr><td>K875004</td><td>LB</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0929" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/29</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder/2</td><td></td></tr> | ||
+ | <tr><td>2</td><td>9/29 PCR INP-His-scFv/2</td><td></td></tr> | ||
+ | <tr><td>3</td><td>9/29 PCR INP-His-scFv/2</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/3e/T--Kyoto--9_29_No157_Electrophoresis_Miyazaki_10_06_04_22_37.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0929" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/29</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/29 PCR INP-His-scFv</td><td>31.9</td><td>2.02</td><td>1.38</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="0930" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>9/30</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>BclA clos/4.0</td><td></td></tr> | ||
+ | <tr><td>3</td><td>INP clos/5.5</td><td></td></tr> | ||
+ | <tr><td>4</td><td>BclA cex/4.5</td><td></td></tr> | ||
+ | <tr><td>5</td><td>INP cex/2.5</td><td></td></tr> | ||
+ | <tr><td>6</td><td>BclA scFv/4.5</td><td></td></tr> | ||
+ | <tr><td>7</td><td>INP scFv/4.0</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>RNA purification</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>OD</th></tr> | ||
+ | <tr><td>BclAcex</td><td>0.504</td></tr> | ||
+ | <tr><td>BclAscFv</td><td>0.788</td></tr> | ||
+ | <tr><td>INPscFv</td><td>0.837</td></tr> | ||
+ | <tr><td>INPclos</td><td>1.564</td></tr> | ||
+ | <tr><td>BclAclos</td><td>0.817</td></tr> | ||
+ | <tr><td>INPcex</td><td>0.839</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/d/d1/T--Kyoto--10_1_No161_General_Miyazaki_10_11_10_14_22.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>RNA extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample</th><th>conc(ng/µl)</th><th>260/280</th><th>260/230</th><th>diluted solution volume(200ng/µl)</th></tr> | ||
+ | <tr><td>1</td><td>INPclos</td><td>359.2</td><td>2.07</td><td>1.21</td><td>11.94</td></tr> | ||
+ | <tr><td>2</td><td>BclAcex</td><td>230.9</td><td>2.03</td><td>0.85</td><td>2.31</td></tr> | ||
+ | <tr><td>3</td><td>INPcex</td><td>1069.0</td><td>2.11</td><td>1.43</td><td>65.17</td></tr> | ||
+ | <tr><td>4</td><td>INPscFv</td><td>320.0</td><td>2.10</td><td>0.93</td><td>9</td></tr> | ||
+ | <tr><td>5</td><td>BclAclos</td><td>386.2</td><td>2.04</td><td>0.97</td><td>13.97</td></tr> | ||
+ | <tr><td>6</td><td>BclAcex</td><td>441.0</td><td>2.04</td><td>0.83</td><td>18</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Reverse Transcription</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>name</th><th>copy(high or low)</th><th>RT</th></tr> | ||
+ | <tr><td>3 nega</td><td>high copy</td><td>-</td></tr> | ||
+ | <tr><td>3 posi</td><td>high copy</td><td>+</td></tr> | ||
+ | <tr><td>4 nega</td><td>low copy</td><td>-</td></tr> | ||
+ | <tr><td>4 posi</td><td>low copy</td><td>+</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>10/1 RT×20 3RT- /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>10/1 RT×20 3RT+ /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>10/1 RT×20 4RT- /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>10/1 RT×20 4RT+ /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>9/27 RT×1 A-6 /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>9/27 RT×1 A-5 /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>9/27 RT×1 B-6 /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | <tr><td>9/27 RT×1 B-5 /1.0µl</td><td>rRNA16s forward primer1 /0.4µl</td><td>rRNA16s reverse primer1 /0.4µl</td><td>2×KAPA HiFi /5µl</td><td>3.2µl</td><td></td><td>10µl</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>10/1 RT×20 3RT- /1.0</td><td>INP primer for CBD /0.4</td><td>CBD cex primer Rv /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>10/1 RT×20 3RT+ /1.0</td><td>INP primer for CBD /0.4</td><td>CBD cex primer Rv /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>10/1 RT×20 4RT- /1.0</td><td>scFv primer1 /0.4</td><td>scFv primer2 /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>10/1 RT×20 4RT+ /1.0</td><td>scFv primer1 /0.4</td><td>scFv primer2 /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>9/27 RT×1 A-6 /1.0</td><td>INP primer for CBD /0.4</td><td>CBD cex primer Rv /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>9/27 RT×1 A-5 /1.0</td><td>INP primer for CBD /0.4</td><td>CBD cex primer Rv /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>9/27 RT×1 B-6 /1.0</td><td>scFv primer1 /0.4</td><td>scFv primer2 /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | <tr><td>9/27 RT×1 B-5 /1.0</td><td>scFv primer1 /0.4</td><td>scFv primer2 /0.4</td><td>2×KAPA HiFi /5</td><td>3.2</td><td></td><td>10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>3RT- target /10</td><td></td></tr> | ||
+ | <tr><td>2</td><td>3RT+ target /10</td><td></td></tr> | ||
+ | <tr><td>3</td><td>3RT- 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>4</td><td>3RT+ 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>5</td><td>4RT- target /10</td><td></td></tr> | ||
+ | <tr><td>6</td><td>4RT+ target /10</td><td></td></tr> | ||
+ | <tr><td>7</td><td>4RT- 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>8</td><td>4RT+ 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>9</td><td>100bp ladder /5</td><td></td></tr> | ||
+ | <tr><td>10</td><td>A-6 target /10</td><td></td></tr> | ||
+ | <tr><td>11</td><td>A-5 target /10</td><td></td></tr> | ||
+ | <tr><td>12</td><td>A-6 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>13</td><td>A-5 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>14</td><td>B-6 target /10</td><td></td></tr> | ||
+ | <tr><td>15</td><td>B-5 target /10</td><td></td></tr> | ||
+ | <tr><td>16</td><td>B-6 16s rRNA /10</td><td></td></tr> | ||
+ | <tr><td>17</td><td>B-5 16s rRNA /10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4a/T--Kyoto--10_1_No165_Electrophoresis_Miyazaki_10_10_07_19_39.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1001" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/1</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>K165002</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>BclA scFv</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>INP scFv</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>BclA cex</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>INP cex</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>BclA clos</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>INP clos</td><td></td><td>DH5α</td><td></td></tr> | ||
+ | <tr><td>K165002</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>BclA scFv</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>INP scFv</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>BclA cex</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>INP cex</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>BclA clos</td><td></td><td>BL21</td><td></td></tr> | ||
+ | <tr><td>INP clos</td><td></td><td>BL21</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1004" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/4</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>INP CTL 8/4 miniprep</td><td></td><td>DH5α/20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1005" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>INP CTL 10/4 transformation</td><td>LB(CP)/200ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1005" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/5</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker(Wako) 5µl</td></tr> | ||
+ | <tr><td>1</td><td>BBa_E165002(NC) </td></tr> | ||
+ | <tr><td>2</td><td>BclA-6His-scFv (33 kDa) </td></tr> | ||
+ | <tr><td>3</td><td>INPNC-6His-scFv (63 kDa) </td></tr> | ||
+ | <tr><td>4</td><td>BclA-6His-CBDcex (17 kDa) </td></tr> | ||
+ | <tr><td>5</td><td>INPNC-6His-CBDcex(47kDa) </td></tr> | ||
+ | <tr><td>6</td><td>BclA-6His-CBDclos (17 kDa) </td></tr> | ||
+ | <tr><td>7</td><td>INPNC-6His-CBDclos (47 kDa) </td></tr> | ||
+ | <tr><td>8</td><td>BBa_K875004(PC) Lpp-OmpA-scFv-6His (43 kDa)</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/cb/T--Kyoto--10_5_No193_General_Miyazaki_10_17_07_20_04.png"> | ||
+ | </div> | ||
+ | <p class="keln_note">Used pelet from 400 µl overnight culture.<br><br>Transfar ;80 min 200 mA(CC)<br>Blocking ;O/N<br>1’antibody ;anti-6His(mouse)<br>1:1000 with blocking buffer 1h<br>2’antibody ;anti-mouse HRP 1:2500 with TBST 1h<br>Detection time 240 sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1006" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>10/5 liquid culture INP CTL /2.5ml</td><td>LB(CP) /47.5ml</td></tr> | ||
+ | <tr><td>10/5 liquid culture INP CTL /2.5ml</td><td>LB(CP) /47.5ml</td></tr> | ||
+ | <tr><td>10/5 liquid culture INP CBD cex(5) /2.5ml</td><td>LB(CP) /47.5ml</td></tr> | ||
+ | <tr><td>10/5 liquid culture INP CBD cex(5) /2.5ml</td><td>LB(CP) /47.5ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1006" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/6</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>10/5 liquid culture INP CTL /100ml</td><td>LB(CP) /100ml</td></tr> | ||
+ | <tr><td>10/3 liquid culture INP CBD cex /100ml</td><td>LB(CP) /200ml</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1007" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/7</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD 16th</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>before</th><th>after</th><th>wash</th></tr> | ||
+ | <tr><td>INPcex+</td><td>0.208/0.208/0.211</td><td>0.224/0.221/0.226</td><td>0.013/0.012/0.010</td></tr> | ||
+ | <tr><td>INPcex-</td><td>0.210/0.216/0.204</td><td>0.232/0.231/0.232</td><td></td></tr> | ||
+ | <tr><td>INPctl+</td><td>0.196/0.196/0.191</td><td>0.220/0.217/0.212</td><td>0.023/0.024/0.025</td></tr> | ||
+ | <tr><td>INPctl-</td><td>0.194/0.195/0.196</td><td>0.207/0.199/0.208</td><td></td></tr> | ||
+ | <tr><td>cellulose only</td><td>0.015/0.013/0.015</td><td>0.031/0.031/0.032</td><td>0.018/0.017/0.019</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1011" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/11</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>2/25 miniprep INPcex</td><td>3</td><td>DH5α /20</td><td>LB(CP)×3</td></tr> | ||
+ | <tr><td>2/8 miniprep INP His 12 (INPscFv)</td><td>3</td><td>DH5α /20</td><td>LB(CP)×3</td></tr> | ||
+ | <tr><td>2/25 miniprep INPcex</td><td>3</td><td>BL21 /20</td><td>LB(CP)×3</td></tr> | ||
+ | <tr><td>2/8 miniprep INP His 12 (INPscFv)</td><td>3</td><td>BL21 /20</td><td>LB(CP)×3</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1011" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/11</h3></span> | ||
+ | <span class="keln_exp"><h4>SEM(CBD)</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample/ml</th><th>before</th><th>after</th><th>wash1</th><th>wash2</th></tr> | ||
+ | <tr><td>INP CBDcex 10/1 liquid culture /90</td><td>0.550</td><td>0.392</td><td>0.074/0.084</td><td>-0.004/-0.018</td></tr> | ||
+ | <tr><td>INP CBDcex 10/1 liquid culture /45</td><td>0.550</td><td>0.432</td><td>0.002/0.000</td><td>-0.001/0.000</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1012" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony1</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony2</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony3</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony4</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony5</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony6</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony7</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony8</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony9</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony10</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony11</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony12</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony13</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony14</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony15</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony16</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony17</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony18</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony19</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony20</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1012" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony21</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony21</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony21</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-scFv-INP-His-CBDcex DH5α colony21</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/8 miniprep INP-His-scFv /0.5</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | <tr><td>2/25 miniprep INP-His-CBDcex /0.5</td><td>VF/0.5 VR/0.5</td><td>11.5</td><td>Quick Taq/12.5</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1012" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/12</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony1 /5</td><td></td></tr> | ||
+ | <tr><td>2</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony2 /5</td><td></td></tr> | ||
+ | <tr><td>3</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony3 /5</td><td></td></tr> | ||
+ | <tr><td>4</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony4 /5</td><td></td></tr> | ||
+ | <tr><td>5</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony5 /5</td><td></td></tr> | ||
+ | <tr><td>6</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony6 /5</td><td></td></tr> | ||
+ | <tr><td>7</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony7 /5</td><td></td></tr> | ||
+ | <tr><td>8</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony8 /5</td><td></td></tr> | ||
+ | <tr><td>9</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony17 /5</td><td></td></tr> | ||
+ | <tr><td>10</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony18 /5</td><td></td></tr> | ||
+ | <tr><td>11</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony19 /5</td><td></td></tr> | ||
+ | <tr><td>12</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony20 /5</td><td></td></tr> | ||
+ | <tr><td>13</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony21 /5</td><td></td></tr> | ||
+ | <tr><td>14</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony22 /5</td><td></td></tr> | ||
+ | <tr><td>15</td><td>INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony23 /5</td><td></td></tr> | ||
+ | <tr><td>16</td><td>INPNC-His-CBDcex 10/12 colony PCR /5</td><td></td></tr> | ||
+ | <tr><td>17</td><td>1kbp ladder /10</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/11/T--Kyoto--10_12_No172_Electrophoresis_Miyazaki_10_15_05_41_02.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1012" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/12</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 INP-His 12 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/8 INP-His 12 /0.1</td><td>VF /0.75</td><td>VR /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample /µl</th><th>primer /µl</th><th>KAPA Taq Extra Hotstart /µl</th><th>DMSO /µl</th><th>MilliQ /µl</th><th>total /µl</th></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony2</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony7</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony17</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony19</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony9</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony10</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony11</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony12</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony13</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony16</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony23</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony24</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony25</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony26</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony27</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony28</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony29</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony30</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony31</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample /µl</th><th>primer /µl</th><th>KAPA Taq Extra Hotstart /µl</th><th>DMSO /µl</th><th>MilliQ /µl</th><th>total /µl</th></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony31</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony32</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony33</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex + INP-scFv colony34</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-scFv /0.25</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | <tr><td>INP-cex /0.25</td><td>VF /1.25 VR /1.25</td><td>12.5</td><td>0.75</td><td>9.25</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/6 liquid culture INP-His-CBDcex</td><td>27.8</td><td>1.96</td><td>1.86</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/12 PCR INP-His 12 (INP-His coding)</td><td>727.3</td><td>1.88</td><td>1.86</td></tr> | ||
+ | <tr><td>10/13 PCR INP-His 12</td><td>1132.1</td><td>1.89</td><td>2.04</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>10/13 PCR purification INP-His coding</td><td>7</td><td>2</td><td>0</td><td>0</td><td>2</td><td>10×H /6</td><td>0</td><td>44</td><td>60</td></tr> | ||
+ | <tr><td>10/13 PCR purification INP-His 12</td><td>6</td><td>0</td><td>2</td><td>0</td><td>2</td><td>10×M /6</td><td>0</td><td>44</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>2</td><td>0</td><td>0</td><td>2</td><td>10×H /6</td><td>0</td><td>0</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>0</td><td>0</td><td>2</td><td>2</td><td>10×H /6</td><td>0</td><td>0</td><td>60</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>INPcex+INPscFv colony2</td><td></td></tr> | ||
+ | <tr><td>2</td><td>INPcex+INPscFv colony7</td><td></td></tr> | ||
+ | <tr><td>3</td><td>INPcex+INPscFv colony19</td><td></td></tr> | ||
+ | <tr><td>4</td><td>INPcex+INPscFv colony9</td><td></td></tr> | ||
+ | <tr><td>5</td><td>INPcex+INPscFv colony17</td><td></td></tr> | ||
+ | <tr><td>6</td><td>INPcex+INPscFv colony10</td><td></td></tr> | ||
+ | <tr><td>7</td><td>INPcex+INPscFv colony11</td><td></td></tr> | ||
+ | <tr><td>8</td><td>INPcex+INPscFv colony12</td><td></td></tr> | ||
+ | <tr><td>9</td><td>INPcex+INPscFv colony15</td><td></td></tr> | ||
+ | <tr><td>10</td><td>INPcex+INPscFv colony16</td><td></td></tr> | ||
+ | <tr><td>11</td><td>INPcex+INPscFv colony23</td><td></td></tr> | ||
+ | <tr><td>12</td><td>INPcex+INPscFv colony24</td><td></td></tr> | ||
+ | <tr><td>13</td><td>INPcex+INPscFv colony25</td><td></td></tr> | ||
+ | <tr><td>14</td><td>INPcex+INPscFv colony26</td><td></td></tr> | ||
+ | <tr><td>15</td><td>INPscFv /5</td><td></td></tr> | ||
+ | <tr><td>16</td><td>INPcex /5</td><td></td></tr> | ||
+ | <tr><td>17</td><td>1kbp ladder</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/15/T--Kyoto--10_13_No178_Electrophoresis_Miyazaki_10_16_03_04_04.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>A1</td><td>INPcex+INPscFv colony2</td><td></td></tr> | ||
+ | <tr><td>A2</td><td>INPcex+INPscFv colony7</td><td></td></tr> | ||
+ | <tr><td>A3</td><td>INPcex+INPscFv colony19</td><td></td></tr> | ||
+ | <tr><td>A4</td><td>INPcex+INPscFv colony9</td><td></td></tr> | ||
+ | <tr><td>A5</td><td>INPcex+INPscFv colony17</td><td></td></tr> | ||
+ | <tr><td>A6</td><td>INPscFv /5</td><td></td></tr> | ||
+ | <tr><td>A7</td><td>INPcex /5</td><td></td></tr> | ||
+ | <tr><td>A8</td><td>1kbp ladder /5</td><td></td></tr> | ||
+ | <tr><td>B1</td><td>INPcex+INPscFv colony10</td><td></td></tr> | ||
+ | <tr><td>B2</td><td>INPcex+INPscFv colony11</td><td></td></tr> | ||
+ | <tr><td>B3</td><td>INPcex+INPscFv colony12</td><td></td></tr> | ||
+ | <tr><td>B4</td><td>INPcex+INPscFv colony13</td><td></td></tr> | ||
+ | <tr><td>B5</td><td>INPcex+INPscFv colony14</td><td></td></tr> | ||
+ | <tr><td>B6</td><td>1kbp ladder /5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/26/T--Kyoto--10_13_No179_Electrophoresis_Miyazaki_10_16_03_20_40.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>10/13 1A</td><td></td></tr> | ||
+ | <tr><td>3</td><td>10/13 2A</td><td></td></tr> | ||
+ | <tr><td>4</td><td>10/13 3A</td><td></td></tr> | ||
+ | <tr><td>5</td><td>10/13 4A</td><td></td></tr> | ||
+ | <tr><td>6</td><td>10/13 5A</td><td></td></tr> | ||
+ | <tr><td>7</td><td>10/13 1B</td><td></td></tr> | ||
+ | <tr><td>8</td><td>10/13 2B</td><td></td></tr> | ||
+ | <tr><td>9</td><td>10/13 3B</td><td></td></tr> | ||
+ | <tr><td>10</td><td>10/13 4B</td><td></td></tr> | ||
+ | <tr><td>11</td><td>10/15 5B</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e2/T--Kyoto--10_13_No180_Electrophoresis_Miyazaki_10_16_03_25_22.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>1A+2A</td><td>10/13 PCR purification INP His coding</td><td></td></tr> | ||
+ | <tr><td>3A~5A</td><td>10/13 midiprep INP His CBDcex</td><td></td></tr> | ||
+ | <tr><td>1B+2B</td><td>10/13 PCR purification INP His12</td><td></td></tr> | ||
+ | <tr><td>3B~5B</td><td>10/13 midiprep INP His CBDcex</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/05/T--Kyoto--10_13_No181_Gel_Extraction_Miyazaki_10_16_03_33_03.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>number</th><th>magnification</th></tr> | ||
+ | <tr><td>INPclos</td><td>13318</td><td>×200</td></tr> | ||
+ | <tr><td>INPclos</td><td>13319~13328</td><td>×600</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13329</td><td>×200</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13330~13338</td><td>×600</td></tr> | ||
+ | <tr><td>cellulose only</td><td>13339</td><td>×200</td></tr> | ||
+ | <tr><td>cellulose only</td><td>13340,13343~13351</td><td>×600</td></tr> | ||
+ | <tr><td>cellulose only</td><td>13341,13342</td><td>×1000</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">without filter</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>E.coli</th><th>OD600</th><th>cellulose</th><th>weight</th><th>filter</th></tr> | ||
+ | <tr><td>-</td><td>-</td><td>powder</td><td>10</td><td>non-filtered</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>0.2</td><td>powder</td><td>10</td><td>non-filtered</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>0.2</td><td>powder</td><td>10</td><td>non-filtered</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>powder</td><td>10</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>0.2</td><td>powder</td><td>10</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>0.2</td><td>powder</td><td>10</td><td>filtered</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>number</th><th>magnification</th></tr> | ||
+ | <tr><td>INPcex</td><td>13353</td><td>×200</td></tr> | ||
+ | <tr><td>INPcex</td><td>13354~13356,13364,13366</td><td>×600</td></tr> | ||
+ | <tr><td>INPcex</td><td>13357,13365</td><td>×1000</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13367</td><td>×200</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13368~13378</td><td>×600</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13379</td><td>×1000</td></tr> | ||
+ | <tr><td>cellulose only</td><td>13380,13381</td><td>×200</td></tr> | ||
+ | <tr><td>cellulose only</td><td>13382~13391</td><td>×600</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">with filter</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1013" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/13</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>number</th><th>magnification</th></tr> | ||
+ | <tr><td>INPcex</td><td>13392~98</td><td>×200</td></tr> | ||
+ | <tr><td>INPcex</td><td>13399~13408</td><td>×600</td></tr> | ||
+ | <tr><td>INP CTL</td><td>13309~13419</td><td>×200</td></tr> | ||
+ | <tr><td>bemliese</td><td>13420~13430</td><td>×200</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">with bemliese</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder /3</td><td></td></tr> | ||
+ | <tr><td>2</td><td>10/13 1A /25</td><td></td></tr> | ||
+ | <tr><td>4</td><td>10/13 2A /25</td><td></td></tr> | ||
+ | <tr><td>5</td><td>10/13 3A /25</td><td></td></tr> | ||
+ | <tr><td>6</td><td>10/13 4A /25</td><td></td></tr> | ||
+ | <tr><td>7</td><td>10/13 5A /25</td><td></td></tr> | ||
+ | <tr><td>8</td><td>10/13 1B /25</td><td></td></tr> | ||
+ | <tr><td>9</td><td>10/13 2B /25</td><td></td></tr> | ||
+ | <tr><td>10</td><td>10/13 3B /25</td><td></td></tr> | ||
+ | <tr><td>11</td><td>10/13 4B /25</td><td></td></tr> | ||
+ | <tr><td>12</td><td>10/13 5B /25</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b9/T--Kyoto--10_14_No186_Electrophoresis_Miyazaki_10_16_04_07_57.1.jpg"><img src="https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--10_14_No186_Electrophoresis_Miyazaki_10_16_04_07_57.2.jpg"> | ||
+ | </div> | ||
+ | <p class="keln_note">with bemliese</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>SEM(CBD)</h4></span> | ||
+ | <span class="keln_researcher">Miyazaki</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>membrane shape</th></tr> | ||
+ | <tr><td>INPcex + cellulose</td><td>square</td></tr> | ||
+ | <tr><td>INP CTL + cellulose</td><td>triangle</td></tr> | ||
+ | <tr><td>cellulose only</td><td>pentagon</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>10/14 gel extract e2 /5</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a0/T--Kyoto--10_14_No188_Electrophoresis_Miyazaki_10_16_06_42_15.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>10/14 electrophoresis 1B longer + 2B longer e1</td><td>10/13 restriction enzyme design INP His12</td><td></td></tr> | ||
+ | <tr><td>10/14 electrophoresis 1B longer + 2B longer e2</td><td>10/13 restriction enzyme design INP His12</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kbp ladder /3</td><td></td></tr> | ||
+ | <tr><td>2</td><td>1A + 2A /3</td><td></td></tr> | ||
+ | <tr><td>3</td><td>3A + 4A + 5A /3</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/7c/T--Kyoto--10_14_No189_Electrophoresis_Miyazaki_10_16_07_13_08.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Gel Extraction</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>Restriction Enzyme Digestion Product</th><th>Volume/(µl)</th></tr> | ||
+ | <tr><td>10/14 electrophoresis 1B + 2B</td><td>10/13 restriction enzyme design INP His12</td><td></td></tr> | ||
+ | <tr><td>10/14 electrophoresis 1A + 2A</td><td>10/13 restriction enzyme design INP His coding</td><td></td></tr> | ||
+ | <tr><td>10/14 electrophoresis 3A + 4A + 5A</td><td>INP His CBDcex</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample1 1A + 2A /µl</th><th>sample2 3A~5A /µl</th><th>Ligation high ver.2</th><th>total</th></tr> | ||
+ | <tr><td>5</td><td>2.5</td><td>7.5</td><td>15</td></tr> | ||
+ | <tr><td>3.75</td><td>3.75</td><td>7.5</td><td>15</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1014" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/14</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>INP His coding ligationA</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP His coding ligationA</td><td>4</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP His coding ligationB</td><td>2</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>INP His coding ligationB</td><td>4</td><td>DH5α /20</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/15 INP-His coding i</td><td>207.4</td><td>1.87</td><td>2.11</td></tr> | ||
+ | <tr><td>10/15 INP-His coding G</td><td>174.7</td><td>1.88</td><td>1.09</td></tr> | ||
+ | <tr><td>10/15 INP-His coding E</td><td>175.5</td><td>1.08</td><td>2.10</td></tr> | ||
+ | <tr><td>10/15 INP-His coding M</td><td>212.9</td><td>1.88</td><td>2.14</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD Binding Assay</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>Buffer</th><th>OD</th></tr> | ||
+ | <tr><td>INP cex</td><td>CBC buffer</td><td>1.002</td></tr> | ||
+ | <tr><td>simultaneous expression</td><td>CBC buffer</td><td>0.994</td></tr> | ||
+ | <tr><td>INP CTL</td><td>CBC buffer</td><td>1.004</td></tr> | ||
+ | <tr><td>INP CTL</td><td>PBS</td><td>0.964</td></tr> | ||
+ | <tr><td>simultaneous expression</td><td>PBS</td><td>1.048</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Restriction Enzyme Digestion</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample</th><th>DNA/(µl)</th><th>EcoR1/(µl)</th><th>Xba1/(µl)</th><th>Spe1/(µl)</th><th>Pst1/(µl)</th><th>10xH Buffer/(µl)</th><th>BSA/(µl)</th><th>MilliQ/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>10/15 PCR purification INP His coding i</td><td>29.0</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>21</td><td>60</td></tr> | ||
+ | <tr><td>10/15 PCR purification INP His coding G</td><td>34.3</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>15.7</td><td>60</td></tr> | ||
+ | <tr><td>10/15 PCR purification INP His coding E</td><td>34.2</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>15.8</td><td>60</td></tr> | ||
+ | <tr><td>10/15 PCR purification INP His coding M</td><td>28.2(6000ng)</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>21.8</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>0</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>0</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>0</td><td>60</td></tr> | ||
+ | <tr><td>10/13 midiprep INP-His-CBDcex</td><td>50</td><td>2</td><td>0</td><td>0</td><td>2</td><td>6</td><td></td><td>0</td><td>60</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">1.5 Dr.Kitabatake<br>2.6 Agarose S + Promega Gel Extraction<br>3.7 Agarose S + Dr.Nasuda<br>4.8 stock</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Single Colony Isolation</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>plate</th></tr> | ||
+ | <tr><td>INPNc-His-scFv </td><td>LB(CP)×3</td></tr> | ||
+ | <tr><td>INPNc-His-CBDcex</td><td>LB(CP)×3</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | <tr><td>2/22 INP-His scFv colony3 /0.1</td><td>INP His coding Fw /0.75</td><td>INP His coding Rv /0.75</td><td>2×KAPA /12.5</td><td>10.9</td><td>0</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>A1 10/14 INPcex + INPscFv colony1</td><td>VF/1.25 VR/1.25</td><td>8.75</td><td>KAPA2G /12.5 DMSO/1.25</td><td>25</td></tr> | ||
+ | <tr><td>A2 10/14 INPcex + INPscFv colony2</td><td>VF/1.25 VR/1.25</td><td>8.75</td><td>KAPA2G /12.5 DMSO/1.25</td><td>25</td></tr> | ||
+ | <tr><td>A3 10/14 INPcex + INPscFv colony3</td><td>VF/1.25 VR/1.25</td><td>8.75</td><td>KAPA2G /12.5 DMSO/1.25</td><td>25</td></tr> | ||
+ | <tr><td>A4 10/14 INPcex + INPscFv colony4</td><td>VF/1.25 VR/1.25</td><td>8.75</td><td>KAPA2G /12.5 DMSO/1.25</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1015" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/15</h3></span> | ||
+ | <span class="keln_exp"><h4>Western Blotting</h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>lane</th><th>sample</th></tr> | ||
+ | <tr><td>Marker</td><td>M.Marker</td></tr> | ||
+ | <tr><td>1</td><td>BBa_K875004(PC) Lpp-OmpA-scFv-6His 43kDa </td></tr> | ||
+ | <tr><td>2</td><td>INPNC-6His-CBDcex 47kDa </td></tr> | ||
+ | <tr><td>3</td><td>INPNC-6His-scFv 63kDa </td></tr> | ||
+ | <tr><td>4</td><td>INPNC-6His-CBDcex/INPNC-6His-scFv</td></tr> | ||
+ | <tr><td>5</td><td>BBa_K165002(NC) </td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/03/T--Kyoto--10_15_No194_General_Miyazaki_10_17_08_24_33.png"> | ||
+ | </div> | ||
+ | <p class="keln_note">Used pelet from 500 µl overnight culture, adjested OD600 to 3.1 .<br><br>Transfar ;80 min 200 mA(CC)<br>Blocking ;O/N<br>1’antibody ;anti-6His(mouse)<br>1:1000 with blocking buffer 1h<br>2’antibody ;anti-mouse HRP 1:2500 with TBST 1h<br>Detection time 900 sec<br>(ECL Prime)</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1kb ladder</td><td></td><td></td></tr> | ||
+ | <tr><td>10/15 colony PCR colony 1</td><td>3</td><td></td></tr> | ||
+ | <tr><td>10/15 colony PCR colony 2</td><td>25</td><td></td></tr> | ||
+ | <tr><td>10/15 colony PCR colony 3</td><td>25</td><td></td></tr> | ||
+ | <tr><td>10/15 colony PCR colony 4</td><td>25</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Cellulose binding Assay</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Cellulose/(mg)</th><th>E.coli/(mg)</th><th>buffer</th></tr> | ||
+ | <tr><td>1</td><td>cex/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>1</td><td>ctl/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>0.5</td><td>cex/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>0.5</td><td>ctl/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>0.5</td><td>CBC/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>0.1</td><td>cex/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | <tr><td>0.1</td><td>ctl/1</td><td>carbonate-bicarbonate buffer</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">RT,-3h 500rpm 15:45~21:15 binding<br>fix with 4×1/100 PFA 10min→centrifuge→PBS→centrifuge<br>→PBS→centrifuge→PBS→centrifuge→DAP1→centrifuge<br>→PBS→centrifuge→PBS→through filter→PBS into filter<br>→centrifuge→PBSinto filter→centrifuge<br>→suspend with PBS→centrifuge<br>→suspend with?discoloration inhibitor<br></p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Dual Display Assay for Electron Microscope</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>E.coli</th><th>Cellulose</th><th>VLP</th><th>buffer</th></tr> | ||
+ | <tr><td>A.Dual 1mL</td><td>1mg</td><td>10µL</td><td>PBS</td></tr> | ||
+ | <tr><td>B.Dual 1mL</td><td>1mg</td><td></td><td>PBS</td></tr> | ||
+ | <tr><td>C.Dual 1mL</td><td></td><td>10µL</td><td>PBS</td></tr> | ||
+ | <tr><td>D.Dual 1mL</td><td></td><td></td><td>PBS</td></tr> | ||
+ | <tr><td>E.INPctl</td><td>1mg</td><td>10µL</td><td>PBS</td></tr> | ||
+ | <tr><td>F.INPctl</td><td>1mg</td><td></td><td>PBS</td></tr> | ||
+ | <tr><td>G.INPctl</td><td></td><td>10µL</td><td>PBS</td></tr> | ||
+ | <tr><td>H.INPctl</td><td></td><td></td><td>PBS</td></tr> | ||
+ | <tr><td>I</td><td>1mg</td><td></td><td>PBS</td></tr> | ||
+ | <tr><td>J</td><td></td><td>10µL</td><td>PBS</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">RT,ih incubate,850rpm<br>Dul=1.020<br>ctl=1.056</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1 kb ladder</td><td>5</td></tr> | ||
+ | <tr><td>2</td><td>10/15 Digestion INP His Coding 2</td><td>30 /0.94 上0.04下0.10</td></tr> | ||
+ | <tr><td>3</td><td>10/15 Digestion INP His Coding 2</td><td>30 /0.94 上0.04下0.19</td></tr> | ||
+ | <tr><td>4</td><td>10/13 miniprep INP-His-CBDcex 6</td><td>30 /0.94 0.11</td></tr> | ||
+ | <tr><td>5</td><td>10/13 miniprep INP-His-CBDcex 6</td><td>30 /0.94</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">Uchino 16:08~<br>ABEF→centrifuge→resupend with PBS →centrifuge→through filter→resupend with PBS→through filter→centrifuge→respend with PBS→centrifuge→resupend with PBS→centrifuge→resupend with PBS<br>IJ Michimori</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>10/15 colony PCR colony1</td><td></td></tr> | ||
+ | <tr><td>3</td><td>10/15 colony PCR colony2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>10/15 colony PCR colony3</td><td></td></tr> | ||
+ | <tr><td>5</td><td>10/15 colony PCR colony</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/ef/T--Kyoto--10_16_No8_Electrophoresis_Sassa_10_17_12_16_22.1.jpg"><img src="https://static.igem.org/mediawiki/2016/a/a2/T--Kyoto--10_16_No8_Electrophoresis_Sassa_10_17_12_16_22.2.jpg | ||
+ | "> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Target)</h4></span> | ||
+ | <span class="keln_researcher">Sassa</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primer1/(µl)</th><th>Primer2/(µl)</th><th>Buffer/(µl)</th><th>MilliQ/(µl)</th><th>Any Other/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>2/22 scFv col.3 0.1</td><td>VF 0.75</td><td>VR 0.75</td><td>KAPA HoFi 12.5</td><td>10.9</td><td></td><td>25</td></tr> | ||
+ | <tr><td>2/22 scFv col.3 0.1</td><td>VF 0.75</td><td>VR 0.75</td><td>KAPA HoFi 12.5</td><td>10.9</td><td></td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">95℃ 5min<br>98℃ 20s×35<br>55℃ 15s ×35<br>72℃ 30s×35<br>4℃ infinity</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino, Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/15 Gel Extraction INP His coding 長</td><td>9.6</td><td>1.88</td><td>0.27</td></tr> | ||
+ | <tr><td>10/15 Gel Extraction INP His coding 短</td><td>74.4</td><td>1.87</td><td>0.84</td></tr> | ||
+ | <tr><td>10/15 Gel Extraction INP His CBDcex</td><td>22.0</td><td>1.97</td><td>0.33</td></tr> | ||
+ | <tr><td>Insert MK</td><td>64.5</td><td>1.92</td><td>1.17</td></tr> | ||
+ | <tr><td>Vector MK</td><td>56.6</td><td>2.37</td><td>0.10</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Ligation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Insert</th><th>Vector</th><th>MilliQ</th><th>Ligation High</th></tr> | ||
+ | <tr><td>10/15 Insert MK/2µL</td><td>10/15 Gel Extract INP His CBDcex 5.5µl</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Insert MK/1µL</td><td>10/15 Gel Extract INP His CBDcex 6µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Insert MK/1µL</td><td>10/15 Gel Extract INP His CBDcex 0.5µL</td><td>6</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding/5µL</td><td>10/15 Digestion INP His CBDcex 2.5µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding/6µL</td><td>10/15 Digestion INP His CBDcex 1.5µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding/4.5µL</td><td>10/15 Digestion INP His CBDcex 3µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/14Gel Extract INP His coding/5.5</td><td>10/15 Digestion INP His CBDcex 2µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/14Gel Extract INP His coding/6</td><td>10/15 Digestion INP His CBDcex 1.5µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding 3.5µL</td><td>10/15 Digestion INP His CBDcex 4µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding 2.5µL</td><td>10/15 Digestion INP His CBDcex 5µL</td><td>0</td><td>7.5</td></tr> | ||
+ | <tr><td>10/15 Digestion INP His coding 3.75</td><td>10/15 Digestion INP His CBDcex 3.75</td><td>0</td><td>7.5</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Transformation</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name</th><th>Sample Volume (µl)</th><th>Competent Cells/(µl)</th><th>Medium</th></tr> | ||
+ | <tr><td>10/15Ligation Insert MK+Gel Extract1 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Insert MK+Gel Extract2 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion3 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion4 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion5 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion6 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Gel Extract+Digestion7 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Gel Extract+Digestion8 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion9 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion10 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/15Ligation Digestion+Digestion11 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/14 Ligation PuroductA 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | <tr><td>10/14 Ligation PuroductB 2µL</td><td>2</td><td>DH5α/2</td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>PCR (Purification)</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Sample Name/(µl)</th><th>Concentration(ng/µl)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/15 PCR scFv INP His codingFw+INP His RV</td><td>284.4</td><td>1.88</td><td>2.18</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>10/14 Tranceformation INPHis coding2 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>10/14 Tranceformation INPHis coding2 colony2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>non-woven felt </h4></span> | ||
+ | <span class="keln_researcher">Ohara</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>null</th></tr> | ||
+ | <tr><td>cex</td><td>13431×200</td></tr> | ||
+ | <tr><td>cex</td><td>32~41×600</td></tr> | ||
+ | <tr><td>ctl</td><td>13442×200</td></tr> | ||
+ | <tr><td>ctl</td><td>43~55×600</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>10/14 Transformation INP His coding 2 colony1</td><td>LB(CP)</td></tr> | ||
+ | <tr><td>10/14 Transformation INP His coding 2 colony2</td><td>LB(CP)</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>fluorescence microscope assay</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>number</th><th>magnification</th></tr> | ||
+ | <tr><td>cex bemliese</td><td>13431</td><td>×200</td></tr> | ||
+ | <tr><td>cex bemliese</td><td>13432~13441</td><td>×600</td></tr> | ||
+ | <tr><td>CTL bemliese</td><td>13442</td><td>×200</td></tr> | ||
+ | <tr><td>CTL bemliese</td><td>13443~13355</td><td>×600</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR</h4></span> | ||
+ | <span class="keln_researcher">Matsumoto, Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>number</th><th>sample</th><th>Any others</th><th>Any others(µL)</th></tr> | ||
+ | <tr><td>1~5</td><td>10/15Ligation Insert MK+Gel Extract1</td><td>2×Ready Mix</td><td>12.5</td></tr> | ||
+ | <tr><td>6~10</td><td>10/15Ligation Insert MK+Gel Extract2</td><td>Fwd primer</td><td>1.25</td></tr> | ||
+ | <tr><td>11~15</td><td>10/15Ligation Insert MK+Gel Extract3</td><td>Rev primer</td><td>1.25</td></tr> | ||
+ | <tr><td>16~20</td><td>10/15Ligation Digestion+Digestion4</td><td>MilliQ</td><td>10</td></tr> | ||
+ | <tr><td>21~24</td><td>10/15Ligation Digestion+Digestion5</td><td></td><td>52×mix</td></tr> | ||
+ | <tr><td>25~28</td><td>10/15Ligation Digestion+Digestion6</td><td>2×Ready</td><td>650</td></tr> | ||
+ | <tr><td>29~32</td><td>10/15Ligation Gel Extract+Digestion7</td><td>Fwd </td><td>65</td></tr> | ||
+ | <tr><td>33~36</td><td>10/15Ligation Gel Extract+Digestion8</td><td>Rev</td><td>65</td></tr> | ||
+ | <tr><td>37~40</td><td>10/15Ligation Digestion+Digestion9</td><td>MilliQ</td><td>500</td></tr> | ||
+ | <tr><td>41~44</td><td>10/15Ligation Digestion+Digestion10</td><td></td><td></td></tr> | ||
+ | <tr><td>45~48</td><td>10/15Ligation Digestion+Digestion11</td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note"> ×16<br>16Colony Picking DMSO 0.75 12.75 12<br> Mix 12.5 212.5 200<br> Fwd 1.25 21.25 18.75 20<br> Rev 1.25 21.25 18.75 20<br> MilliQ 9.25 157.25 138.75 148<br>16sample colony1~14・・・P15 sample colony<br>Blue 15・・・2/8 INP-His9<br> 16・・・8/1 miniprep INPcex colony</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>1</td><td></td></tr> | ||
+ | <tr><td>3</td><td>2</td><td></td></tr> | ||
+ | <tr><td>4</td><td>3</td><td></td></tr> | ||
+ | <tr><td>5</td><td>4</td><td></td></tr> | ||
+ | <tr><td>6</td><td>5</td><td></td></tr> | ||
+ | <tr><td>7</td><td>6</td><td></td></tr> | ||
+ | <tr><td>8</td><td>7</td><td></td></tr> | ||
+ | <tr><td>9</td><td>8</td><td></td></tr> | ||
+ | <tr><td>10</td><td>9</td><td></td></tr> | ||
+ | <tr><td>11</td><td>10</td><td></td></tr> | ||
+ | <tr><td>12</td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4d/T--Kyoto--10_16_No21_Electrophoresis_Sassa_10_17_10_54_10.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td></td><td></td></tr> | ||
+ | <tr><td>2</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>3</td><td>11</td><td></td></tr> | ||
+ | <tr><td>4</td><td>12</td><td></td></tr> | ||
+ | <tr><td>5</td><td>13</td><td></td></tr> | ||
+ | <tr><td>6</td><td>14</td><td></td></tr> | ||
+ | <tr><td>7</td><td>15</td><td></td></tr> | ||
+ | <tr><td>8</td><td>16</td><td></td></tr> | ||
+ | <tr><td>9</td><td>17</td><td></td></tr> | ||
+ | <tr><td>10</td><td>18</td><td></td></tr> | ||
+ | <tr><td>11</td><td>19</td><td></td></tr> | ||
+ | <tr><td>12</td><td>20</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/76/T--Kyoto--10_16_No21_Electrophoresis_Sassa_10_17_10_54_18.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>E.coli</th><th>OD600</th><th>cellulose</th><th>size / weight</th><th>filter</th></tr> | ||
+ | <tr><td>-</td><td>-</td><td>bemliese</td><td>5mm×5mm</td><td>-</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>1</td><td>bemliese</td><td>5mm×5mm</td><td>-</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>1</td><td>bemliese</td><td>5mm×5mm</td><td>-</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>powder</td><td>1</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>1</td><td>powder</td><td>1</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>1</td><td>powder</td><td>1</td><td>filtered</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>powder</td><td>0.5</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>1</td><td>powder</td><td>0.5</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>1</td><td>powder</td><td>0.5</td><td>filtered</td></tr> | ||
+ | <tr><td>-</td><td>-</td><td>powder</td><td>0.1</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-ctl</td><td>1</td><td>powder</td><td>0.1</td><td>filtered</td></tr> | ||
+ | <tr><td>INPNC-His-CBDcex</td><td>1</td><td>powder</td><td>0.1</td><td>filtered</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1016" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/16</h3></span> | ||
+ | <span class="keln_exp"><h4>fluorescence microscope assay</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>sample</th><th>number</th><th>magnification</th></tr> | ||
+ | <tr><td>cex 1mg</td><td>13456</td><td>×200</td></tr> | ||
+ | <tr><td>cex 1mg</td><td>13457~13466</td><td>×600</td></tr> | ||
+ | <tr><td>cex 0.5mg</td><td>13467</td><td>×200</td></tr> | ||
+ | <tr><td>cex 0.5mg</td><td>13468~13378</td><td>×600</td></tr> | ||
+ | <tr><td>cex 0.5mg</td><td>13379</td><td>×1000</td></tr> | ||
+ | <tr><td>CTL 1mg</td><td>13380</td><td>×200</td></tr> | ||
+ | <tr><td>CTL 1mg</td><td>13381~13390</td><td>×600</td></tr> | ||
+ | <tr><td>CTL 0.5mg</td><td>13391</td><td>×200</td></tr> | ||
+ | <tr><td>CTL 0.5mg</td><td>13392~13401</td><td>×600</td></tr> | ||
+ | <tr><td>CTL 0.5mg</td><td>13402</td><td>×1000</td></tr> | ||
+ | <tr><td>cex 0.1mg</td><td>13403</td><td>×200</td></tr> | ||
+ | <tr><td>cex 0.1mg</td><td>13404,13407~13416</td><td>×600</td></tr> | ||
+ | <tr><td>cex 0.1mg</td><td>13405,13406</td><td>×1000</td></tr> | ||
+ | <tr><td>CTL 0.1mg</td><td>13417</td><td>×200</td></tr> | ||
+ | <tr><td>CTL 0.1mg</td><td>13418~13430</td><td>×600</td></tr> | ||
+ | <tr><td>CTL 0.1mg</td><td>13431</td><td>×200</td></tr> | ||
+ | <tr><td>cellulose 0.5mg</td><td>13432~13441</td><td>×600</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>21</td><td></td></tr> | ||
+ | <tr><td>2</td><td>22</td><td></td></tr> | ||
+ | <tr><td>3</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>4</td><td>23</td><td></td></tr> | ||
+ | <tr><td>5</td><td>24</td><td></td></tr> | ||
+ | <tr><td>6</td><td>25</td><td></td></tr> | ||
+ | <tr><td>7</td><td>26</td><td></td></tr> | ||
+ | <tr><td>8</td><td>27</td><td></td></tr> | ||
+ | <tr><td>9</td><td>28</td><td></td></tr> | ||
+ | <tr><td>10</td><td>29</td><td></td></tr> | ||
+ | <tr><td>11</td><td>30</td><td></td></tr> | ||
+ | <tr><td>12</td><td></td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--10_17_No22_Electrophoresis_Sassa_10_17_07_25_05.1.jpg"><img src="https://static.igem.org/mediawiki/2016/9/96/T--Kyoto--10_17_No22_Electrophoresis_Sassa_10_17_07_25_05.2.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>31</td><td></td></tr> | ||
+ | <tr><td>3</td><td>32</td><td></td></tr> | ||
+ | <tr><td>4</td><td>33</td><td></td></tr> | ||
+ | <tr><td>5</td><td>34</td><td></td></tr> | ||
+ | <tr><td>6</td><td>35</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/13/T--Kyoto--10_17_No23_Electrophoresis_Sassa_10_17_08_53_22.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher"></span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>36</td><td></td></tr> | ||
+ | <tr><td>2</td><td>37</td><td></td></tr> | ||
+ | <tr><td>3</td><td>38</td><td></td></tr> | ||
+ | <tr><td>4</td><td>39</td><td></td></tr> | ||
+ | <tr><td>5</td><td>40</td><td></td></tr> | ||
+ | <tr><td>6</td><td>1kb ladder</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/ba/T--Kyoto--10_17_No23_Electrophoresis_Sassa_10_17_08_54_39.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>2</td><td>41</td><td></td></tr> | ||
+ | <tr><td>3</td><td>42</td><td></td></tr> | ||
+ | <tr><td>4</td><td>43</td><td></td></tr> | ||
+ | <tr><td>5</td><td>44</td><td></td></tr> | ||
+ | <tr><td>6</td><td>45</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--10_17_No24_Electrophoresis_Sassa_10_17_08_57_02.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Electrophoresis</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Lane</th><th>sample/(µl)</th><th>Length(bp)</th></tr> | ||
+ | <tr><td>1</td><td></td><td></td></tr> | ||
+ | <tr><td>2</td><td>1kb ladder</td><td></td></tr> | ||
+ | <tr><td>3</td><td>45</td><td></td></tr> | ||
+ | <tr><td>4</td><td>46</td><td></td></tr> | ||
+ | <tr><td>5</td><td>47</td><td></td></tr> | ||
+ | <tr><td>6</td><td>48</td><td></td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/2b/T--Kyoto--10_17_No24_Electrophoresis_Sassa_10_17_08_57_47.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/ea/T--Kyoto--10_17_No26_Colony_PCR_%28typeA%29_Sassa_10_17_09_47_35.jpg"> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Liquid Culture</h4></span> | ||
+ | <span class="keln_researcher">Yamamoto</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Name</th><th>medium</th></tr> | ||
+ | <tr><td>colony 1,2,3,4,5,11,12,13,14,15,18,19,22,24,26,30</td><td><LB(CP)/td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">total 16mL</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Miniprep</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>DNA</th><th>Concentration/(µg/ml)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>1</td><td>65.8</td><td>1.91</td><td>2.02</td></tr> | ||
+ | <tr><td>2</td><td>63.2</td><td>1.90</td><td>1.84</td></tr> | ||
+ | <tr><td>3</td><td>84.6</td><td>1.87</td><td>1.33</td></tr> | ||
+ | <tr><td>4</td><td>100.8</td><td>1.92</td><td>1.67</td></tr> | ||
+ | <tr><td>5</td><td>84.6</td><td>1.89</td><td>1.59</td></tr> | ||
+ | <tr><td>11</td><td>89.3</td><td>1.84</td><td>1.43</td></tr> | ||
+ | <tr><td>12</td><td>87.3</td><td>1.92</td><td>1.88</td></tr> | ||
+ | <tr><td>13</td><td>88.0</td><td>1.89</td><td>1.59</td></tr> | ||
+ | <tr><td>14</td><td>79.8</td><td>1.91</td><td>1.72</td></tr> | ||
+ | <tr><td>15</td><td>83.3</td><td>1.90</td><td>1.82</td></tr> | ||
+ | <tr><td>18</td><td>102.5</td><td>1.88</td><td>1.50</td></tr> | ||
+ | <tr><td>19</td><td>114.9</td><td>1.91</td><td>1.59</td></tr> | ||
+ | <tr><td>22</td><td>109.9</td><td>1.78</td><td>1.14</td></tr> | ||
+ | <tr><td>24</td><td>80.4</td><td>1.93</td><td>2.00</td></tr> | ||
+ | <tr><td>26</td><td>64.6</td><td>1.93</td><td>1.84</td></tr> | ||
+ | <tr><td>30</td><td>88.8</td><td>1.80</td><td>1.07</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">total 16mL</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>Colony PCR (typeA)</h4></span> | ||
+ | <span class="keln_researcher">Yamada</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Templates/(µl)</th><th>Primers/(µl)</th><th>MilliQ/(µl)</th><th>PreMix/(µl)</th><th>Total/(µl)</th></tr> | ||
+ | <tr><td>10/15gel plated colony 1</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>10/15gel plated colony</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>INPNC-His-scFv</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | <tr><td>INP-His-CBDcex</td><td>VF1.25 VR1.25</td><td>9.25</td><td>KapaTag/12.5 DMSO 0.75</td><td>25</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p class="keln_note">VF 1.25×8=10µL<br>VR 1.25×8=10µL<br>Kapa 12.5×8=100µL<br>DM80 0.75×8=6µL<br>MilliQ 9.25×8=74µL<br>Total 200µL</p> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Sassa --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1017" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/17</h3></span> | ||
+ | <span class="keln_exp"><h4>CBD Binding Assay</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>Dual 1mL OD=1.0</th><th>bemlise 5mm×5mm</th><th>VLP Narita104</th></tr> | ||
+ | <tr><td>Dual 1mL OD=1.0</td><td>bemlise 5mm×5mm</td><td>10µL</td></tr> | ||
+ | <tr><td>ctll 1mL OD=1.0</td><td>bemlise 5mm×5mm</td><td>10µL</td></tr> | ||
+ | <tr><td>ctl 1mL OD=1.0</td><td>bemlise 5mm×5mm</td><td>10µL</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
+ | <!-- Table Generated by KELN Author: Miyazaki --> | ||
+ | <div class="keln_container"> | ||
+ | <a name="1018" class = "kyoto-jump"></a> | ||
+ | <span class="keln_date"><h3>10/18</h3></span> | ||
+ | <span class="keln_exp"><h4>Fluorescence Microscopy</h4></span> | ||
+ | <span class="keln_researcher">Uchino</span> | ||
+ | <table class="keln_table"> | ||
+ | <tr><th>E.coli</th><th>OD600</th><th>cellulose</th><th>size</th><th>filter</th></tr> | ||
+ | <tr><td>-</td><td>-</td><td>bemliese</td><td>5mm×5mm</td><td>-</td></tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!------------ Table END ------------> | ||
Latest revision as of 16:52, 19 October 2016
![](https://static.igem.org/mediawiki/2016/7/76/T--Kyoto--pagephoto-01.png)
August | ||||||
---|---|---|---|---|---|---|
1 | ||||||
2 | 3 | 4 | 5 | 6 | 7 | 8 |
9 | 10 | 11 | 12 | 13 | 14 | 15 |
16 | 17 | 18 | 19 | 20 | 21 | 22 |
23 | 24 | 25 | 26 | 27 | 28 | 29 |
30 | 31 |
September | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | ||
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | |||
8/14
Transformation
Li, Uchino, KanekoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
RFP 0.5ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 5ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 10ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 20ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 50ng/µl | 2 | DH5α /20 | LB(CP) |
8/14
Gel Extraction
Li, Uchino, KanekoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 10/1 PQ(3) | about 23.5 |
2 | 1kb ladder | 6 |
3 | 10/1 PQ(3) | about 23.5 |
4 | 10/1 PQ(3) | about 24 |
![](https://static.igem.org/mediawiki/2016/7/7a/T--Kyoto--8_14_No1_Gel_Extraction_Notsu_09_19_12_14_09No.20814.jpg)
Total volume of samples of lane 1~3 was 71.5µl and it was divided into three.
8/14
Transformation
Li, Uchino, KanekoSample Name | Sample Volume | Competent Cells/(µl) | Medium |
---|---|---|---|
3C5 | 10µl | DH5α 20µl | LB(CP) |
P+ | 10µl | DH5α 20µl | LB(CP) |
P- | 10µl | DH5α 20µl | LB(CP) |
RBS | 10µl | DH5α 20µl | LB(CP) |
8/14
Liquid Culture
Li, Uchino, Iguchi, KanekoName | medium |
---|---|
8/13 transformation RFP 20ng/µl colony1 | LB(CP) |
8/13 transformation part RFP colony1 | LB(CP) |
8/13 transformation part RFP colony2 | LB(CP) |
8/13 transformation part RFP colony3 | LB(CP) |
8/13 transformation part RFP colony4 | LB(CP) |
8/13 transformation part RFP colony5 | LB(CP) |
8/13 transformation part RFP colony6 | LB(CP) |
8/13 transformation part RFP colony7 | LB(CP) |
8/13 transformation part RFP colony8 | LB(CP) |
8/13 transformation part RFP colony9 | LB(CP) |
8/13 transformation part RFP colony10 | LB(CP) |
8/13 transformation part RFP colony11 | LB(CP) |
8/15
Miniprep
Li, Uchino, Notsu, Kaneko, WatanabeDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
RFP1 /2µl | 82.5 | 1.40 | 1.22 |
RFP2 /4µl | 142.5 | 1.69 | 2.05 |
RFP3 /4µl | 50 | 1.55 | 1.71 |
RFP4 /4µl | 57.5 | 1.64 | 1.88 |
RFP6 /4µl | 98.75 | 1.71 | 1.40 |
RFP(20) /4µl | 76.25 | 1.67 | 1.32 |
8/15
Electrophoresis
Notsu, KanekoLane | Restriction Enzyme Digestion Products |
---|---|
1 | 1kb ladder 3µl |
2 | RFP1 10µl |
3 | RFP2 10µl |
4 | RFP3 10µl |
5 | RFP4 10µl |
6 | RFP6 10µl |
7 | RFP(20) 10µl |
8 | PCR1 5µl |
9 | PCR2 5µl |
10 | PCR3 5µl |
11 | PCR4 5µl |
12 | PCR5 5 µl |
13 | 1kb ladder 3µl |
8/15
Restriction Enzyme Digestion
Uchino, Notsu, KanekoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/15Miniprep PSB1C3-RFP1 | 24 | 1 | 0 | 0 | 1 | 5(H) | 0 | 19 | 50 |
8/15Miniprep PSB1C3-RFP2 | 14 | 1 | 0 | 0 | 1 | 5(H) | 0 | 29 | 50 |
8/15Miniprep PSB1C3-RFP3 | 40 | 1 | 0 | 0 | 1 | 5(H) | 0 | 3 | 50 |
8/15Miniprep PSB1C3-RFP4 | 35 | 1 | 0 | 0 | 1 | 5(H) | 0 | 8 | 50 |
8/15Miniprep PSB1C3-RFP6 | 20 | 1 | 0 | 0 | 1 | 5(H) | 0 | 23 | 50 |
8/15
colony PCR
Uchino, Notsu, Kaneko, Watanabecolony Primers |
---|
8/14 transformation pSB1C3-RFP1 VF+VR |
8/14 transformation pSB1C3-RFP2 VF+VR |
8/14 transformation pSB1C3-RFP3 VF+VR |
8/14 transformation pSB1C3-RFP4 VF+VR |
8/14 transformation pSB1C3-RFP1 VF+VR |
8/15
PCR (Steps)
Uchino, Notsu, Kaneko, WatanabeSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 94℃ | 2min | 1 |
Denature | 94℃ | 30sec | 30 |
Annealing | 55℃ | 30sec | 30 |
Extension | 68℃ | 1min | 30 |
Final elongation | 4℃ | 7min | 1 |
Final Holding | 4℃ | - | - |
8/16
Gel Extraction
Li, Iguchi, Notsu, Kaneko, WatanabeLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 10 |
2 | blank | |
3 | 8/15 digestion pSB1C3-RFP 1 | 55 |
4 | 8/15 digestion pSB1C3-RFP 2 | 55 |
5 | 8/15 digestion pSB1C3-RFP 3 | 36 |
6 | 8/15 digestion pSB1C3-RFP 4 | 36 |
7 | 8/15 digestion pSB1C3-RFP 6 | 36 |
![](https://static.igem.org/mediawiki/2016/7/77/T--Kyoto--8_16_No10_Gel_Extraction_Notsu_09_26_01_32_24.jpg)
8/16
Electrophoresis
Li, Iguchi, Notsu, Kaneko, WatanabeLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 8/16 Gel Extraction RFP1 /1 | |
2 | 8/16 Gel Extraction RFP2 /1 | |
3 | 8/16 Gel Extraction RFP3 /1 | |
4 | 8/16 Gel Extraction RFP4 /1 | |
5 | blank | |
6 | 8/16 pSB1C3-vector /1 | |
7 | 8/16 pSB1C3-vector /1 | |
8 | 1kladder |
![](https://static.igem.org/mediawiki/2016/6/6f/T--Kyoto--8_16_No11_Electrophoresis_Notsu_09_26_01_55_23.jpg)
8/16
Ligation
Li, Iguchi, Notsu, Kaneko, WatanabeVector DNA /(µl) | insert DNA /(µl) | ligase /(µl) | 10×buffer /(µl) | MilliQ /(µl) | Total /(µl) |
---|---|---|---|---|---|
pSB1C3 EPcut /1 | RFP /4 | 0.5 | 1 | 3 | 9.5 |
pSB1C3 EPcut /1 | RFP /2 | 0.5 | 1 | 5 | 9.5 |
pSB1C3 EPcut /1 | RFP /4 | 0.5 | 1 | 3 | 9.5 |
pSB1C3 EPcut /1 | RFP /4 | 0.5 | 1 | 3 | 9.5 |
pSB1C3 EPcut /1 | - | 0.5 | 1 | 7 | 9.5 |
8/17
Liquid Culture
LiName | medium |
---|---|
8/16 transformation pSB3C5 RFP1 | LB(CP) /6 |
8/16 transformation pSB3C5 RFP2 | LB(CP) /6 |
8/16 transformation pSB3C5 RFP1 | LB(CP) /6 |
8/17
Transformation
LiSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
pSB1C3 P+ | 2 | DH5α /20 | LB |
pSB1C3 P- | 2 | DH5α /20 | LB |
pSB1C3 RBS | 2 | DH5α /20 | LB |
pSB3C5 RFP | 2 | DH5α /20 | LB |
8/16 ligation pSB1C3 RFP1 | 2 | DH5α | LB |
8/17
Electrophoresis
IguchiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kbp ladder /3 | |
2 | blank | |
3 | pSB1C3-RFP(ligate)1 /2 | |
4 | pSB1C3-RFP(ligate)2 /2 | |
5 | pSB1C3-RFP(ligate)3 /2 | |
6 | pSB1C3-RFP(ligate)4 /3 | |
7 | Vector (Ctrl) /5 |
8/18
Liquid Culture
Li, Iguchi, NotsuName | medium |
---|---|
INP [1] BBa_K811005 | LB(CP) / 6ml |
INP [2] BBa_K811005 | LB(CP) / 6ml |
INP [3] BBa_K811005 | LB(CP) / 6ml |
scfv04 [1] BBa_K875004 | LB(CP) / 6ml |
scfv04 [2] BBa_K875004 | LB(CP) / 6ml |
scfv04 [3] BBa_K875004 | LB(CP) / 6ml |
scfv06 [1] BBa__K875006 | LB(CP) / 6ml |
scfv06 [2] BBa_K875006 | LB(CP) / 6ml |
scfv06 [3] BBa_K875006 | LB(CP) / 6ml |
8/18
Miniprep
Li, Iguchi, NotsuDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/17 Liquid Culture 3C5 [1] / 5µl | 77 | 1.69 | 1.45 |
8/17 Liquid Culture 3C5 [2] / 2µl | 40 | 1.67 | 1.17 |
8/17 Liquid Culture 3C5 [3] / 2µl | 15 | 3.75 | 3.70 |
8/18
Electrophoresis
LiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /1 | - |
2 | [1] psb3c5(8/18 miniprep) /4.2 | 3807 |
3 | [2] psb3c5(8/18 miniprep) /4.2 | 3807 |
4 | [1'] psb3c5(8/18 miniprep) /4.2 | 3807 |
5 | [2'] psb3c5(8/18 miniprep) /4.2 | 3807 |
6 | [3'] psb3c5(8/18 miniprep) /4.2 | 3807 |
![](https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--8_18_No18_Electrophoresis_Kaneko_05_20_04_12_53.jpg)
[img18]
8/18
Restriction Enzyme Digestion
Li, Iguchi, NotsuSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
psb3c5(8/18miniprep) [1'], [2'], [3'] mixed / 50 | 1.0 | 1.0 | 5.6 | 56.6 |
8/19
Transformation
Li, Iguchi, NotsuSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
BBa_ J61002 p+ | 2 | DH5a /20 | LB(Amp) |
BBa_J61002 p- | 2 | DH5a /20 | LB(Amp) |
BBa_1A2 RBS | 2 | DH5a /20 | LB(Amp) |
Failed due to deactivation of Amp
8/19
MutationPCR
Templates /µl | Primer /µl | Primer STAR Max Premix /µl | MilliQ /µl | Total(µl) |
---|---|---|---|---|
scfv plasmid /0.1 | m.scfv FW /1, m.scfvRV/1 | 25 | 22.9 | 50 |
scfv plasmid (nega) /0.1 | 0 | 5 | 4.9 | 10 |
(nega) /0 | m.scfv FW/0.2, m.scfvRV/0.2 | 5 | 4.6 | 10 |
---30cycle---
98℃ 10''
55℃ 15''
72℃ 20''
----------------
4℃ holding
8/19
Electrophoresis
NotsuLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/2 | - |
2 | scfv plasmid +primer(m.scfv FW, m.scfv RV) mutation PCR product /5 | 3600 |
3 | scfv plasmid mutation PCR product /5 | - |
4 | primer(m.scfv FW, m.scfv RV) mutation PCR product /5 | - |
5 | psb3c5 EPcut product /5 | 2600(backborn), 1200(inseert |
![](https://static.igem.org/mediawiki/2016/6/64/T--Kyoto--8_19_No22_Electrophoresis_Kaneko_05_20_04_27_06.jpg)
[img22]
8/19
Concentration
Li, NotsuSample | OD600 | Bacterial cells /ml |
---|---|---|
INP [1] K811005 | 0.643 | 5.14x10^8 |
scfv06 K875006 | 0.634 | 5.07x10^8 |
scfv04 K875004 | 0.618 | 4.94x10^8 |
1 OD 600 equals to 8x10^8 cells /ml
8/19
Miniprep
Li, NotsuDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
scfv 04[1] K875004 /3µl | 56 | 1.63 | 1.45 |
scfv 04[2] K875004 /3µl | 56 | 1.63 | 1.45 |
scfv 04[3] K875004 /3µl | 56 | 1.63 | 1.45 |
scfv 06[1] K875006 /3µl | 56 | 1.63 | 1.45 |
scfv 06[2] K875006 /3µl | 56 | 1.63 | 1.45 |
scfv 06[3] K875006 /3µl | 56 | 1.63 | 1.45 |
INP [1] K811005 /3µl | 56 | 1.63 | 1.45 |
INP [2] K811005 /3µl | 56 | 1.63 | 1.45 |
INP [3] K811005 /3µl | 56 | 1.63 | 1.45 |
There was a problem with an instrument
8/20
Transformation
Li, UchinoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/19 mutation PCR scfv plasmid (nega) | 1 | DH5a/ 20 | LB(CP) |
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.
8/20
Transformation
Li, UchinoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/19 mutation PCR scfv plasmid | 1 [note:1] | DH5a/20 | LB(AMP) |
8/19 mutation PCR scfv plasmid (nega) | 1 [note:2] | DH5a/20 | LB(AMP) |
BBa_J61002 p+ | 1 | DH5a/20 | LB(AMP) |
BBa_J61002 p- | 1 | DH5a/20 | LB(AMP) |
BBa_1A2 RBS | 1 | DH5a/20 | LB(AMP) |
[note:1]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/50µl was folded 300 times.
[note:2]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.
8/20
Electrophoresis
LiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /1.5 | |
2 | 9/19 miniprep scfv04 [1] | |
3 | 9/19 miniprep scfv04 [2] | |
4 | 9/19 miniprep scfv04 [3] | |
5 | 9/19 miniprep scfv06 [1] | |
6 | 9/19 miniprep scfv06 [2] | |
7 | 9/19 miniprep scfv06 [3] | |
8 | 9/19 miniprep INP [1] | |
9 | 9/19 miniprep INP [2] | |
10 | 9/19 miniprep INP [3] |
![](https://static.igem.org/mediawiki/2016/5/53/T--Kyoto--8_20_No26_Electrophoresis_Kaneko_05_20_04_44_45.jpg)
[img26]
8/21
Colony PCR
UchinoColony | Primers |
---|---|
8/20 transformation RBS | VF+VR |
8/20 transformation p+ | VF+VR |
8/20 transformation p- | VF+VR |
8/20 dilution INP | VF+VR |
8/21
PCR (Steps)
UchinoSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre | 94℃ | 2min | 1 |
De | 94℃ | 30 sec | 30 |
An | 55℃ | 30 sec | 30 |
Ex | 68℃ | 1min | 30 |
Final E | 4℃ | 7min | 1 |
Final Hold | 4℃ | - | - |
Autoclaved distrilled water /23µl
2xQuick taq /25µl
10 pmol/µl primer (VF) /1µl
10 pmol/µl primer (VR) /1µl
Template DNA /0µl
Total 50µl
8/21
Electrophoresis
LiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /2 | |
2 | 8/21 colony PCR RBS /all | |
3 | 8/21 colony PCR p+ /all | |
4 | 8/21 colony PCR p- /all | |
5 | 8/21 colony PCR INP /all | |
6 | 8/21 colony PCR Nega /all |
![](https://static.igem.org/mediawiki/2016/0/04/T--Kyoto--8_21_No28_Electrophoresis_Kaneko_05_20_04_54_01%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/b/b5/T--Kyoto--8_21_No28_Electrophoresis_Kaneko_05_20_04_54_01%2C2.jpg)
[img29-1]
weak band except INP -> retry
colony PCR and Electrophoresis were conducted under the same condition.
[img29-2]
RBS and INP success.
P+ and P- failed -> retry.
8/21
PCR (Steps)
LiSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 25 |
Annealing | 70℃ | 15sec | 25 |
Extension | 72℃ | 30sec | 25 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | 1 |
8/21
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
scfv 8/20 transformation | cscfv.f /0.75 | cscfv.r /0.75 | 2x KAPA Hifi /12.5 | 11 | 25 | |
- (nega) | cscfv.f /0.3 | cscfv.f /0.3 | 2x KAPA Hifi /5 | 4.4 | 10 |
8/21
Colony PCR
LiColony | Primers |
---|---|
8/20 transformation p+ [1] | VF+VR |
8/20 transformation p+ [2] | VF+VR |
8/20 transformation p+ [3] | VF+VR |
8/20 transformation p- [1] | VF+VR |
8/20 transformation p- [2] | VF+VR |
8/20 transformation p- [3] | VF+VR |
8/18 parts p+ | VF+VR |
8/18 parts p- | VF+VR |
8/21
PCR (Steps)
LiSteps | Temparature | Time | Cycle |
---|---|---|---|
pre | 94℃ | 2min | 1 |
de | 94℃ | 30sec | 30 |
an | 55℃ | 30sec | 30 |
ex | 68℃ | 1min | 30 |
F.elong | 4℃ | 7min | 1 |
F.hold | 4℃ | - | 1 |
8/21
Liquid Culture
LiName | medium |
---|---|
strain | Medium/ml |
8/20 transformation RBS | PG(AMP) /6 |
8/20 dilution INP7 | PG(CP) /6 |
8/17 transformation 3c5, RFP | PG(CP) /6 |
8/20 transformation p+ [1] | PG(AMP) /6 |
8/20 transformation p- [1] | PG(AMP) /6 |
8/21
Electrophoresis
LiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /1.5 | |
2 | 8/21 cloning PCR scfv[1] /2 | |
3 | 8/21 cloning PCR scfv[2] /2 | |
4 | nega /2 | |
5 | 8/21 colony PCR p+ [1] /2 | |
6 | 8/21 colony PCR p+ [2] /2 | |
7 | 8/21 colony PCR p+ [3] /2 | |
8 | 8/21 colony PCR p- [1] /2 | |
9 | 8/21 colony PCR p- [2] /2 | |
10 | 8/21 colony PCR p- [3] /2 | |
11 | 8/21 colony PCR parts p+ /2 | |
12 | 8/21 colony PCR parts p- /2 |
![](https://static.igem.org/mediawiki/2016/9/9d/T--Kyoto--8_21_No34_Electrophoresis_Kaneko_05_20_05_13_01.jpg)
[img35]
2, 3, 4 cloning PCR failed.
1 forgot to put ladder
5, 7, 8, 9 strong band
6 weak
10 failed
P+[1] and p-[1] -> liquid culture
8/24
Ligation
Lisample/µl | MilliQ (µl) | Buffer /µl | Ligase(µl) | Total(µl) |
---|---|---|---|---|
8/19 mutation PCR scfv /5 | 3.5 | 1 | 0.5 | 10 |
8/24
Miniprep
LiDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/20 Liquid culture RBS | 124.5 | 1.65 | 1.02 |
8/20 Liquid culture INP | 147.8 | 1.66 | 0.98 |
8/20 Liquid culture 3c5.RFP | 234.8 | 1.79 | 1.62 |
8/20 Liquid culture P+[1] | 40.3 | 1.78 | 1.60 |
8/20 Liquid culture P-[1] | 56.7 | 1.80 | 2.05 |
8/25
Miniprep
Li, Uchino, Notsu, KanekoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/24 LIquid Culture m.scfv colonyA | - | - | - |
8/24 LIquid Culture m.scfv colonyB | - | - | - |
8/25
Electrophoresis
Lane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /1.5 | |
2 | 8/24 miniprep p+(J61002) /2 | |
3 | 8/24 miniprep p-(J61002) /2 | |
4 | 8/24 miniprep RFP (3c5) /2 | |
5 | 8/24 miniprep INP (1c3) /2 | |
6 | 8/24 miniprep RBS (1A2) /2 |
![](https://static.igem.org/mediawiki/2016/f/fd/T--Kyoto--8_25_No38_Electrophoresis_Kaneko_05_20_05_22_35.jpg)
[img38]
8/25
Restriction Enzyme Digestion
UchinoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
p+/24.8 | 1 | 1 | 10xH /4 | 9.2 | 40 | ||||
IINP /6.8 | 1 | 1 | 10xM /4 | 27.2 | 40 | ||||
p- /17.6 | 1 | 1 | 10xH /4 | 16.4 | 40 | ||||
RBS /8.0 | 1 | 1 | 10xH /4 | 26 | 40 | ||||
RFP /4.3 | 1 | 1 | 10xH /4 | 29.7 | 40 |
8/25
Gel Extraction
Uchino, NotsuLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 6 |
2, 3 | 8/25 digestion p+ | 24 each |
4, 5 | 8/25 digestion p- | 24 each |
6, 7 | 8/25 digestion RFP | 24 each |
8, 9 | 8/25 digestion INP | 24 each |
10, 11 | 8/25 digestion RBS | 24 each |
[img40]
8/25
Actvation check of Xba and Pst
UchinoINP(µl) | Xba(µl) | Pst(µl) | 10xM(µl) | BSA(µl) | MilliQ(µl) | Total(µl) |
---|---|---|---|---|---|---|
0.68 | 0.1 | 0.1 | 1 | 0.2 | 7.92 | 10 |
0.68 | 0.1 | - | 1 | 0.2 | 8.02 | 10 |
0.68 | - | 0.1 | 1 | 0.2 | 8.02 | 10 |
0.68 | - | - | 1 | 0.2 | 8.12 | 10 |
8/25
Electrophoresis
NotsuLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /3 | |
2 | [1] (inp xp cut) /10 | |
3 | [2] (inp x cut) /10 | |
4 | [3] (inp p cut) /10 | |
5 | [4] (inp no cut) /10 |
![](https://static.igem.org/mediawiki/2016/4/46/T--Kyoto--8_25_No41_2_Electrophoresis_Kaneko_05_20_05_34_47.jpg)
[img42]
8/26
Restriction Enzyme Digestion
Uchino, NotsuSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/24 miniprep INP 1c3 /0.68 | - | 1.0 | 0.2 | 7.12 | 10 | ||||
8/15 miniprep RFP psb1c3 /1.3 | - | 1.0 | 0.2 | 6.5 | 10 | ||||
8/24 miniprep INP 1c3 /0.68 | 0.1 | 1.0 | 0.2 | 7.02 | 10 | ||||
8/15 miniprep RFP psb1c3 /1.3 | 0.1 | 1.0 | 0.2 | 6.4 | 10 | ||||
8/24 miniprep INP 1c3/ 0.68 | 0.1 | - | 0.2 | 8.02 | 10 | ||||
8/15 miniprep RFP psb1c3 /1.3 | 0.1 | - | 0.2 | 7.4 | 10 | ||||
8/24 miniprep INP 1c3 /0.68 | - | - | 0.2 | 8.12 | 10 | ||||
8/15 miniprep RFP psb1c3 /1.3 | - | - | 0.2 | 7.5 | 10 |
8/26
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
scfv from MK | c.scfv fw/0.75 | c.scfv rv/0.75 | 2x KAPA Hifi /12.5 | 11 | 25 | |
none | c.scfv fw/0.75 | c.scfv rv/0.75 | 2x KAPA Hifi /5 | 4.4 | 10 |
8/26
Activation Check of Xba
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/2 | |
2 | [8] RFP/10 | |
3 | [6] RFP EcoRI/10 | |
4 | [2] RFP XbaI/10 | |
5 | [4] RFP EcoRI, XbaI /10 | |
6 | [7] INP /10 | |
7 | [5] INP EcoRI /10 | |
8 | [1] INP XbaI /10 | |
9 | [3] INP EcoRI, XbaI/10 |
![](https://static.igem.org/mediawiki/2016/1/17/T--Kyoto--8_26_No45_Electrophoresis_Kaneko_05_20_05_42_54.jpg)
[img45-1]
[img45-2]
8/27
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
s.BAN | BAN.Fw/ 0.75 | BAN.Rv/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 | |
s.BAN | BAN.Fw/ 0.75 | BAN.Rv/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 | |
s.BAN | BAN.Fw/ 0.75 | BAN.Histag/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 | |
scfv | scfv.Fw/ 0.75 | scfv.Rv/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 | |
8/24 miniprep INP | INP.Fw/ 0.75 | INP.Rv/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 | |
8/24 miniprep INP | INP.Fw/ 0.75 | INP.Rv.Histag/ 0.75 | 11 | 2xKAPA HiFi/ 12.5 | 25 |
8/27
PCR (Steps)
LiSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95 | 3min | 1 |
Denature | 98 | 20sec | 25 |
Annealing | 65 | 15sec | 25 |
Extension | 72 | 30sec | 25 |
Final Elogation | 72 | 5min | 1 |
Final Holding | 4 | - | 1 |
9/8
Restriction Enzyme Digestion
NotsuSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
[1] 8/15 miniprep 1c3 RFP /14 | 0.5 | 0.5 | - | 10xH /3 | 12 | 30 | |||
[2] 8/24 miniprep 3c5 RFP /8.5 | 0.5 | - | 0.5 | 10xH /3 | 17.5 | 30 | |||
[3] 8/24 miniprep 3c5 RFP /8.5 | 0.5 | 0.5 | - | 10xH /3 | 17.5 | 30 | |||
[4] 8/24 miniprep BBa_J61002 p- /24 | 0.5 | - | 0.5 | 10xH /3 | 2 | 30 |
![](https://static.igem.org/mediawiki/2016/4/4c/T--Kyoto--9_8_No50_Restriction_Enzyme_Digestion_Kaneko_05_20_05_48_28.jpg)
9/9
Electrophoresis
NotsuLane | Restriction Enzyme Digestion Products |
---|---|
1 | 100bp ladder 9µL |
2 | 8/24 miniprep p- EPcut 30µL |
9/9
Electrophoresis
Sukegawa, NotsuLane | Restriction Enzyme Digestion Products |
---|---|
1 | 1kbp ladder 9µL |
2 | 8/15 miniprep 1C3RFP EScut 30µL |
3 | 8/24 miniprep 3C5RFP EPcut 30µL |
4 | 8/24 miniprep 3C5RFP EScut 30µL |
9/14
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
8/24 miniprep INP | INPfw/0.75 | INPRv/0.75 | 2×KAPAHiFi | 11 | 25 | |
8/24 miniprep INP | INPfw/0.75 | INPHisRv/0.75 | 2×KAPAHiFi | 11 | 25 | |
BAN | BANFw/0/75 | BANRv/0.75 | 2×KAPAHiFi | 11 | 25 | |
BAN | BANFw/0/75 | BANHisRv/0.75 | 2×KAPAHiFi | 11 | 25 | |
BAN | BANFw/0/75 | RBSRv/0.75 | 2×KAPAHiFi | 11 | 25 | |
scFv fromMk | scFvFw/0/75 | scFvRv/0/75 | 2×KAPAHiFi | 11 | 25 | |
scFv fromMk | scFvHisFw/0.75 | scFvRv/0.75 | 2×KAPAHiFi | 11 | 25 |
9/14
PCR (Steps)
LiSteps | Temparature | Time | Cycle |
---|---|---|---|
1 | 95 | 3min | 1 |
2 | 98 | 70sec | 25 |
3 | 68 | 15sec | 25 |
4 | 72 | 30sec | 25 |
5 | 72 | 5min | 1 |
6 | 10 | - | 1 |
9/14
Gel Extraction
KanekoLane | Restriction Enzyme Digestion Products |
---|---|
2 | ladder/6 |
4 | 9/14 PCR1/10 |
6 | 9/14 PCR2/10 |
8 | 9/14 PCR3/10 |
10 | 9/14 PCR4/10 |
12 | 9/14PCR5/10 |
14 | 9/14 PCR6/10 |
16 | 9/14 PCR7/10 |
9/14
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3sBAN/0.15+6scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | 25 | ||
4sBAN/0.15+7scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | 25 | ||
1INP8/24miniprep/0.15+5sBAN0.15+scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | 25 | ||
2INP8/24miniprep/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | 25 |
9/15
Restriction Enzyme Digestion
Li, UchinoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3sBAN+6scFvfromMKPCRproduct | 34 | 1 | 1 | 4 | 40 | ||||
4sBAN+7scFvfromMK PCRproduct | 34 | 1 | 1 | 4 | 40 | ||||
1INP8/24miniprep+5sBAN+6scFvfromMKPCRproduct | 16 | 1 | 1 | 2 | 20 | ||||
2INP8/24miniprep+5sBAN+7scFvfromMkPCRproduct | 34 | 1 | 1 | 4 | 40 |
9/15
Gel Extraction
Li, UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | |
2 | 3sBAN+6scFvfromMkPCRproductEScut | 20 |
3 | 3sBAN+6scFvfromMkPCRproductEScut | 20 |
4 | 4sBAN+7scFvfromMkPCRproductEScut | 20 |
5 | 4sBAN+7scFvfromMkPCRproductEScut | 20 |
6 | 1INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut | 20 |
7 | 2INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut | 20 |
8 | 2INP8/24miniprep+5sBAN+7scFvfromMkPCRproductEScut | 20 |
9 | RFP3C5PCRproduct |
![](https://static.igem.org/mediawiki/2016/a/a2/T--Kyoto--9_15_No56_Gel_Extraction_Yamamoto_08_23_11_46_18%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/5/52/T--Kyoto--9_15_No56_Gel_Extraction_Yamamoto_08_23_11_46_18%2C2.jpg)
9/16
Colony PCR (2Column)
Uchino, MichimoriName I | Name II |
---|---|
9/15 transformation BANscFv 3C5 colony1~16 | VF+VR |
9/15 transformation INPscFv 3C5 colony1~16 | VF+VR |
9/15 transformation BANHisscFv 3C5 colony1~16 | VF+VR |
9/15 transformation INPHisscFv 3C5 colony1~16 | VF+VR |
9/16
PCR (Steps)
Uchino, MichimoriSteps | Temparature | Time | Cycle |
---|---|---|---|
1 | 94 | 2 | 1 |
2 | 94 | 0.5 | 25 |
3 | 55 | 0.5 | 25 |
4 | 68 | 1 | 25 |
5 | 4 | 7 | 1 |
6 | 4 | - | 1 |
9/16
Gel Extraction
Li, Kaneko, NotsuLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | BANscFv3C5 colony 9 | 10 |
3 | BANscFv3C5 colony 10 | 10 |
4 | BANscFv3C5 colony 11 | 10 |
5 | BANscFv3C5 colony 12 | 10 |
6 | BANscFv3C5 colony 13 | 10 |
7 | BANscFv3C5 colony 14 | 10 |
8 | BANscFv3C5 colony 15 | 10 |
9 | BANscFv3C5 colony 16 | 10 |
10 | BANHisscFv3C5 colony 9 | 10 |
11 | BANHisscFv3C5 colony 10 | 10 |
12 | BANHisscFv3C5 colony 11 | 10 |
13 | BANHisscFv3C5 colony 12 | 10 |
14 | BANHisscFv3C5 colony 13 | 10 |
15 | BANHisscFv3C5 colony 14 | 10 |
16 | BANHisscFv3C5 colony 15 | 10 |
17 | BANHisscFv3C5 colony 16 | 10 |
18 | BANscFv3C5 colony 1 | 10 |
9/16
Gel Extraction
Li, Kaneko, NotsuLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | INPscFv3C5 colony 9 | 10 |
3 | INPscFv3C5 colony 10 | 10 |
4 | INPscFv3C5 colony 11 | 10 |
5 | INPscFv3C5 colony 12 | 10 |
6 | INPscFv3C5 colony 13 | 10 |
7 | INPscFv3C5 colony 14 | 10 |
8 | INPscFv3C5 colony 15 | 10 |
9 | INPscFv3C5 colony 16 | 10 |
10 | INPHisscFv3C5 colony 9 | 10 |
11 | INPHisscFv3C5 colony 10 | 10 |
12 | INPHisscFv3C5 colony 11 | 10 |
13 | INPHisscFv3C5 colony 12 | 10 |
14 | INPHisscFv3C5 colony 13 | 10 |
15 | INPHisscFv3C5 colony 14 | 10 |
16 | INPHisscFv3C5 colony 15 | 10 |
17 | INPHisscFv3C5 colony 16 | 10 |
18 | BANscFv3C5 colony 1 | 10 |
![](https://static.igem.org/mediawiki/2016/3/3d/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/8/88/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C2.jpg)
![](https://static.igem.org/mediawiki/2016/4/42/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C3.jpg)
![](https://static.igem.org/mediawiki/2016/6/69/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C4.jpg)
9/17
Electrophoresis
Michimori, KanekoLane | Restriction Enzyme Digestion Products |
---|---|
1 | buffer/5 |
2 | 1kb ladder/5 |
3 | BAN 9 P/4.5 |
4 | BAN 9 N/4.5 |
5 | BAN14 P/4.5 |
6 | BAN14 N/4.5 |
7 | BANHis14 P/4.5 |
8 | BANHis14/4.5 |
![](https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--9_17_No62_Electrophoresis_Yamamoto_08_23_12_42_58.jpg)
9/17
Electrophoresis
KanekoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kbp ladder | |
2 | none | |
3 | BANHis16 p | |
4 | BANHis16 n | |
5 | INP 12 p | |
6 | INP 14 p | |
7 | INP 12 n | |
8 | INP 14 n |
![](https://static.igem.org/mediawiki/2016/9/9b/T--Kyoto--9_17_No63_Electrophoresis_Iguchi_10_17_06_39_35.jpg)
9/17
Electrophoresis
KanekoLane | Restriction Enzyme Digestion Products |
---|---|
1 | - |
2 | - |
3 | 1kbladder/5 |
4 | INPHis9 P/4.5 |
5 | INPHis9N/4.5 |
6 | INPHis12P/4.5 |
7 | INPHis12N/4.5 |
8 | INP14 N/4.5 |
![](https://static.igem.org/mediawiki/2016/b/b8/T--Kyoto--9_17_No64_Electrophoresis_Yamamoto_08_23_01_05_58.jpg)
9/17
Restriction Enzyme Digestion
Uchino, MichimoriSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
BAN9 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
BAN9 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
BAN14 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
BAN14 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
BANHis14 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
BANHis 14 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
BANHis 16 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
BANHis16 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
INP12 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
INP12 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
INP14 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
INP 14 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
INP 9 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
INP9 | 1 | 0 | 0 | 10×H/1 | 8 | 10 | |||
INPHis12 | 2 | 0.5 | 0.5 | 10×H/1 | 6 | 10 | |||
INPHis12 | 1 | 0 | 0 | 10×H/1 | 8 | 10 |
![](https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--9_17_No65_Restriction_Enzyme_Digestion_Yamamoto_08_23_01_13_52.jpg)
9/17
Miniprep
NotsuDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/16 liquid culture BANscFv12 | |||
9/16 liquid culture BANscFv14 | |||
9/16 liquid culture BANHisscFv12 | |||
9/16 liquid culture BANHisscFv14 | |||
9/16 liquid culture INPHisscFv12 | |||
9/16 liquid culture INPHisscFv14 | |||
9/16 liquid culture INPHisscFv10 | |||
9/16 liquid culture BANscFv |
2/4
PartsAwaking
Kaneko, WanDescription | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
---|---|---|---|---|---|---|
RFP | E1010 | pSB1C3 | 11N | 2015 | 3 | 10 |
2/4
Transformation
Uchino, Iguchi, Notsu, WanSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
E1010 | 2 | DH5α | LB(CP) |
2/5
Colony PCR (typeA)
Uchino, Egashira, YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/4 transformation E1010 colony1 | VF 6.25µl VR 6.25µl | 12.5 | 2×Quick Taq HS DyeMix 25µl | 50 |
2/5
PCR (Steps)
Uchino, Egashira, YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 94℃ | 2min | 1 |
Denature | 94℃ | 30sec | 25 |
Annealing | 51℃ | 30sec | 25 |
Extension | 68℃ | 90sec | 25 |
Final Elongation | 4℃ | - | 1 |
Final Holding | 4℃ | - | 1 |
2/5
Transformation
UchinoSample Name | Sample Volume | Competent Cells/(µl) | Medium |
---|---|---|---|
9/17miniprep BAN9 | 2 | DH5α/20µL | |
9/17miniprep BANHis14 | 2 | DH5α/20µL | |
9/17miniprep INPHis12 | 2 | DH5α/20µL | |
9/17miniprep INPHis9 | 2 | DH5α/20µL |
2/5
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 2/5 colony PCR E1010 | 1019 |
3 | other sample | - |
4 | other sample | - |
5 | other sample | - |
6 | other sample | - |
7 | other sample | - |
8 | other sample | - |
![](https://static.igem.org/mediawiki/2016/4/44/T--Kyoto--2_5_No75_Electrophoresis_Notsu_10_01_04_31_28.jpg)
2/6
Miniprep
Notsu, Kaneko, Egashira, Wan, YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/5 liquid culture E1010(RFP) colony1 | 48.0 | 1.93 | 1.42 |
2/6
Restriction Enzyme Digestion
Kaneko, Egashira, Wan, YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/6 miniprep E1010 colony1 | 34 | 0 | 1 | 0 | 1 | 1×M /4 | 0 | 0 | 40 |
2/7
Gel Extraction
Kaneko, YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 10 |
2 | E1010 colony1 2/6 XPcut | 40 |
3 | other sample | - |
4 | other sample | - |
2/7
Gel Extraction(Measurement)
Kaneko, YamamotoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
E1010 colony1 2/6 XPcut | 8.2 | 1.03 | 0.49 |
![](https://static.igem.org/mediawiki/2016/7/71/T--Kyoto--2_7_No79_Gel_Extraction%28Measurement%29_Notsu_10_01_04_40_48.jpg)
![](https://static.igem.org/mediawiki/2016/7/71/T--Kyoto--2_7_No79_Gel_Extraction%28Measurement%29_Notsu_10_01_04_40_48.jpg)
2/7
Liquid Culture
UchinoName | medium |
---|---|
2/5 transformation BAN9 colony1 | LB(CP) |
2/5 transformation BAN-HIS14 colony1 | LB(CP) |
2/5 transformation INP-HIS12 colony1 | LB(CP) |
2/5 transformation BAN9 colony1 | LB(CP) |
2/8
Liquid Culture
Uchino, YamamotoName | medium |
---|---|
2/4 transformation E1010 colony2 1 | LB(CP) |
2/4 transformation E1010 colony2 2 | LB(CP) |
2/8
Miniprep
Uchino, Egashira, YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/7 Liquid culture BAN9 | 123.6 | 1.87 | 2.07 |
2/7 Liquid culture BAN9 | 248.8 | 1.83 | 1.84 |
2/7 Liquid culture BAN-HIS14 | 268.9 | 1.86 | 2.15 |
2/7 Liquid culture BAN-HIS14 | 127.9 | 1.85 | 2.08 |
2/7 Liquid culture INP-HIS9 | 354.3 | 1.86 | 2.24 |
2/7 Liquid culture INP-HIS9 | 289.4 | 1.85 | 1.92 |
2/7 Liquid culture INP-HIS12 | 172.3 | 1.87 | 2.07 |
2/7 Liquid culture INP-HIS12 | 286.5 | 1.86 | 2.20 |
2/8
Electrophoresis
Uchino, Egashira, YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 2/8 miniprep BAN9 /5 | |
3 | 2/8 miniprep BAN9 /5 | |
4 | 2/8 miniprep BAN-HIS14 /5 | |
5 | 2/8 miniprep BAN-HIS14 /5 | |
6 | 2/8 miniprep INP-HIS9 /5 | |
7 | 2/8 miniprep INP-HIS9 /5 | |
8 | 2/8 miniprep INP-HIS14 /5 | |
9 | 2/8 miniprep INP-HIS9 /5 |
![](https://static.igem.org/mediawiki/2016/0/0b/T--Kyoto--2_8_No85_Electrophoresis_Notsu_09_29_01_38_11.jpg)
2/9
Miniprep
Matsumoto, Notsu, Egashira, Yamamoto, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/8 liquid culture E1010 colony2 1and 2 | 58.5 | 1.89 | 2.16 |
2/9
Restriction Enzyme Digestion
Uchino, Egashira, Yamamoto, YamadaSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/8 liquid culture E1010 colony2 | 30 | 0 | 1 | 0 | 1 | 10×M /4 | 2 | 2 | 40 |
2/9
Restriction Enzyme Digestion
Uchino, Yamamoto, YamadaSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/8 liquid culture E1010 colony2 | 5 | 0 | 0 | 0 | 0 | 10×M /2 | 1 | 12 | 20 |
2/10
Electrophoresis
Uchino, Yamamoto, YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 2/9 Negative Control E1010 colony2 /10 | 2776 |
3 | 2/9 Digestion E1010 colony2 p+x /40 | 732 |
4 | 2/9 Negative Control E0040 colony2 /20 | 2799 |
5 | 2/9 Digestion E0040 colony2 p+x /40 | 746 |
6 | 2/9 Negative Control K592009 colony2 /20 | 2739 |
7 | 2/9 Digestion K592009 colony2 p+x /40 | 695 |
8 | 2/9 Negative Control E0022 colony2 /20 | 2832 |
9 | 2/9 Digestion E0022 colony2 p+x /40 | 788 |
2/10
Electrophoresis
Uchino, Yamamoto, YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
3 | 2/9 NC J23105 colony2 /20 | 2105 |
4 | 2/9 Digestion J23105 colony2 E+S /40 | 58 |
5 | 2/9 NC J23100 colony2 /20 | 2105 |
6 | 2/9 Digestion J23100 colony2 E+S /40 | 58 |
7 | 2/9 NC B0034 colony3 /20 | 2091 |
8 | 2/9 Digestion B0034 colony3 P+S /40 | 2080 |
2/10
Miniprep
Michimori, Egashira, Yamamoto, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
E1010 | 9.4 | 2.13 | 0.61 |
K592009 | 5.2 | 1.74 | 0.29 |
E0040 | 15.0 | 1.94 | 0.38 |
E0022 | 8.5 | 1.04 | 0.86 |
B0034 | 21.2 | 1.84 | 1.06 |
Ideal MIniprep effficiency of E1010 would be 18.5ng/microL (presuming 100% Restriction Enzyme Digestion efficiency and 80 % Gel Extraction efficiency).
2/12
PartsAwaking
UchinoDescription | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
---|---|---|---|---|---|---|
sfGFP-CBDcex | K1321342 | pSB1C3 | 18E | 2015 | 5 | 10 |
sfGFP-CBDclos | K1321341 | pSB1C3 | 18C | 2015 | 5 | 10 |
2/12
Transformation
UchinoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/12 parts awakening sfGFP-CBDcex | 1 | DH5α /20 | LB(CP) |
2/12 parts awakening sfGFP-CBDclos | 1 | DH5α /20 | LB(CP) |
2/12
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 miniprep BAN-His14 4 /0.1 | VF /0.75 | m.scFv.Rv /0.75 | - | 10.9 | 2×KAPA HiFi /12.5 | 25 |
2/8 miniprep BAN-His14 4 /0.1 | VR /0.75 | m.scFv.Fw /0.75 | - | 10.9 | 2×KAPA HiFi /12.5 | 25 |
2/12
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 2/12 PCR VF.m.scFv.Rv | 25 |
3 | 2/12 PCR VR.m.Fw | 25 |
![](https://static.igem.org/mediawiki/2016/0/0a/T--Kyoto--2_12_No101_Electrophoresis_Notsu_10_01_07_30_03%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/c/c8/T--Kyoto--2_12_No101_Electrophoresis_Notsu_10_01_07_30_03%2C2.jpg)
2/12
PartsAwaking
UchinoDescription | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
---|---|---|---|---|---|---|
Promoter | J23114 | J61002 | 19J | 2015 | 4 | 10 |
Promoter | J23108 | pSB1C3 | 4C | 2015 | 4 | 10 |
2/12
Transformation
Uchino, YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/12 Parts awakening J23114 | 1 | DH5α /20 | LB(AMP) |
2/12 Parts awakening J23108 | 1 | DH5α /20 | LB(AMP) |
2/12
Gel Extraction(Measurement)
YamadaSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/12 PCR VF.m.scFv.Rv | 18.8 | 1.95 | 1.52 |
2/12 PCR VR.m.scFv.Fw | 28.6 | 2.08 | 0.07 |
2/15
Colony PCR (typeA)
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/12 transformation sfGFP-CBDcex colony1 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDcex colony2 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDclos colony1 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDclos colony2 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/15
Electrophoresis
KanekoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 2/12 transformation sfGFP-CBDcex colony1 | |
3 | 2/12 transformation sfGFP-CBDcex colony2 | |
4 | 2/12 transformation sfGFP-CBDclos colony1 | |
5 | 2/12 transformation sfGFP-CBDclos colony2 |
![](https://static.igem.org/mediawiki/2016/3/32/T--Kyoto--2_15_No3_Electrophoresis_Notsu_10_02_06_38_04.jpg)
2/15
Liquid Culture
Wan, YamamotoName | medium |
---|---|
2/12 transformation sfGFP-CBDcex colony2 | LB(CP) |
2/12 transformation sfGFP-CBDclos colony2 | LB(CP) |
2/15
Colony PCR (typeA)
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/12 transformation J23114 colony1 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23114 colony2 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23108 colony1 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23108 colony2 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/15
PCR (Steps)
UchinoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/15
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35 | VF /0.75 | VR /0.75 | - | 10.12 | 2×KAPA HiFi /12.5 | 25 |
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35 | VF /0.75 | VR /0.75 | - | 10.12 | 2×KAPA HiFi /12.5 | 25 |
2/15
Electrophoresis
KanekoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | other sample | - |
3 | other sample | - |
4 | other sample | - |
5 | other sample | - |
6 | other sample | - |
7 | other sample | - |
8 | other sample | - |
9 | other sample | - |
10 | other sample | - |
11 | other sample | - |
12 | other sample | - |
13 | other sample | - |
14 | 2/12 transformation J23114 colony1 /10 | |
15 | 2/12 transformation J23114 colony2 /10 | |
16 | 2/12 transformation J23108 colony1 /10 | |
17 | 2/12 transformation J23108 colony2 /10 |
2/15
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1ka ladder | 5 |
2 | 2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 25 |
3 | 2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 25 |
2/15
Liquid Culture
Wan, YamadaName | medium |
---|---|
2/12 transformation J23114 colony2 | LB(AMP) |
2/12 transformation J23108 colony2 | LB(AMP) |
2/16
Miniprep
Uchino, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/15 liquid culture sfGFP-CBDcex colony2 | 43.5 | 1.91 | 1.60 |
2/15 liquid culture sfGFP-CBDclos colony2 | 74.0 | 1.86 | 1.82 |
2/16
PCR (Steps)
Steps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/16
PCR (Target)
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/16 miniprep sfGFP-CBDcex colony2 /0.3 | cex.Fw /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16 miniprep sfGFP-CBDclos colony2 /0.3 | cex.Fw /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16
Gel Extraction(Measurement)
YamamotoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 48.6 | 1.83 | 0.19 |
2/16
PCR (Target)
Yamamoto, YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3 | VF /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3 | VF /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16
Miniprep
Uchino, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/15 liquid culture J23114 | 178.8 | 1.91 | 2.20 |
2/16
PCR (Purification)
YamadaSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
2/16 PCR m.scFv | 152.6 | 1.86 | 2.10 |
2/16
Restriction Enzyme Digestion
Matsumoto, Notsu, WanSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/16 PCR m.scFv | 4 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 11 | 30 |
K592009 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
2/16
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | m.scFv 2/16 EPcut | 30 |
3 | K592009 2/16EPcut | 30 |
2/17
PCR (Target)
Uchino, KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 miniprep BAN-His14 /0.1 | BAN-His-cex.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep BAN-His14 /0.1 | BAN-His-clos.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His12 /0.1 | INP-His-cex.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His12 /0.1 | INP-His-clos.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/17
PCR (Steps)
Uchino, KimSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 25 |
Annealing | 65℃ | 15sec | 25 |
Extension | 72℃ | 30sec | 25 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | 1 |
2/17
Gel Extraction
Michimori, Wan, Yamamoto, KimLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | blank | |
2 | 2/17PCR BAN-His-CBDcexRv+VF | 25 |
3 | 2/17PCR BAN-His-CBDclosRv+VF | 25 |
4 | 2/17PCR INP-His-CBDcexRv+VF | 25 |
5 | 2/17PCR INP-His-CBDclosRv+VF | 25 |
6 | 2/16PCR sf-GFP-CBDcex | 50 |
7 | 2/16PCR sf-GFP-CBDclos | 50 |
8 | blank | - |
9 | 1kb ladder | - |
![](https://static.igem.org/mediawiki/2016/2/2c/T--Kyoto--2_17_No8_Gel_Extraction_Notsu_10_02_07_09_31.2..jpg)
2/17
Gel Extraction(Measurement)
Michimori, Wan, Yamamoto, KimSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/17PCR BAN-His-CBDcexRv+VF | 10.0 | 1.86 | 0.43 |
2/17PCR BAN-His-CBDclosRv+VF | 34.6 | 1.91 | 1.81 |
2/17PCR INP-His-CBDcexRv+VF | 100.2 | 1.90 | 1.24 |
2/17PCR INP-His-CBDclosRv+VF | 116.5 | 1.90 | 2.08 |
2/16PCR sf-GFP-CBDcex | 244.0 | 1.91 | 2.08 |
2/16PCR sf-GFP-CBDclos | 208.9 | 1.90 | 2.01 |
2/17
PCR (Target)
YamamotoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/17 Gel Extraction BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex /0.5 | VF /0.75 | VR /0.75 | - | 2 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos /2 | VF /0.75 | VR /0.75 | - | 2 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex /1 | VF /0.75 | VR /0.75 | - | 3.5 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos /2 | VF /0.75 | VR /0.75 | - | 0 | 2×KAPA HiFi /12.5 | 25 |
2/17
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/17
Miniprep
Matsumoto, KimDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/16 liquid culture J23108 colony3 | 20.8 | 1.90 | 1.28 |
2/17
Gel Extraction(Measurement)
Notsu, WanSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/16 Gel Extraction m.scFv EPcut | 14.3 | 1.87 | 1.09 |
2/16 Gel Extraction K592009 EPcut | 14.1 | 1.86 | 1.60 |
2/17
Ligation
Wan, YamamotoVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
2/16 Gel Extraction K592009 EPcut /2.5 | 2/16 Gel Extraction m.scFv EPcut /5.0 | 7.5 | 15 |
2/17
Transformation
Wan, YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/17 ligation m.scFv EPcut+K592009 EPcut | 2 | DH5α /20 | LB(CP) |
2/18
Gel Extraction(Measurement)
KimSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 72.7 | 1.87 | 1.93 |
2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos | 112.1 | 1.92 | 1.34 |
2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 102.4 | 1.89 | 1.96 |
![](https://static.igem.org/mediawiki/2016/a/ac/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_28_40.1.jpg)
![](https://static.igem.org/mediawiki/2016/7/7e/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_28_40.2.jpg)
2/18
Gel Extraction(Measurement)
KimSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos | 103.4 | 1.94 | 2.04 |
![](https://static.igem.org/mediawiki/2016/9/97/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_32_29.1.jpg)
![](https://static.igem.org/mediawiki/2016/3/30/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_32_29.2.jpg)
2/18
Gel Extraction
KimLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 8 |
2 | 2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 25 |
3 | 2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos | 25 |
4 | 2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 25 |
2/18
Gel Extraction
KimLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 8 |
2 | 2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos | 25 |
3 | blank | - |
4 | blank | - |
2/18
Gel Extraction(Measurement)
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
BAN-CBDcex | 72.3 | 1.85 | 1.87 |
BAN-CBDclos | 79.8 | 1.86 | 1.26 |
INP-CBDcex | 83.6 | 1.80 | 1.57 |
INP-CBDclos | 39.4 | 1.76 | 1.77 |
2/18
PCR (Target)
KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
BAN-CBDcex /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
BAN-CBDclos /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
INP-CBDcex /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
INP-CBDclos /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
2/18
Colony PCR (typeA)
UchinoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/17 transformation m.scFv EPcut+K592009 EPcut colony1 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony2 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony3 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony4 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/18
PCR (Steps)
UchinoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 2min | 1 |
Denature | 95℃ | 10sec | 30 |
Annealing | 55℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/18
PCR (Purification)
Michimori, KimSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
2/18 PCR m.scFv EPcut+K592009 EPcut colony1 | 49.8 | 1.92 | 0.08 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony2 | 41.1 | 1.81 | 1.90 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony3 | 47.2 | 1.87 | 1.99 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony4 | 47.5 | 1.87 | 1.93 |
2/19
PCR (Purification)
YamamotoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
BAN-cex/(25) | 161.1 | 1.88 | 2.13 |
BAN-clos/(25) | 217.9 | 1.86 | 2.10 |
INP-cex/(25) | 158.6 | 1.86 | 1.98 |
INP-clos/(25) | 163.6 | 1.85 | 1.70 |
2/19
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/19 purification BAN-cex | 12.4 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.6 | 30 |
2/19 purification BAN-clos | 9.2 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 15.8 | 30 |
2/19 purification INP-cex | 12.6 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.4 | 30 |
2/19 purification INP-clos | 12.2 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.8 | 30 |
2/9 miniprep E1010 | 25 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 0 | 30 |
2/19
Gel Extraction
Yamamoto, Egashira, WanLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 2/19 Reatriction Enzyme Digestion BAN-cex E, Pcut | 30 |
2 | 2/19 Reatriction Enzyme Digestion BAN-clos E, Pcut | 30 |
3 | 2/19 Reatriction Enzyme Digestion INP-cex E, Pcut | 30 |
0 | 1kb ladder | 5 |
![](https://static.igem.org/mediawiki/2016/6/65/T--Kyoto--2_19_No18_Gel_Extraction_Kosaka_09_24_12_03_17.1.jpg)
![](https://static.igem.org/mediawiki/2016/8/80/T--Kyoto--2_19_No18_Gel_Extraction_Kosaka_09_24_12_03_17.2.jpg)
2/19
Gel Extraction
Egashira, Wan, YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 2/19 Restriction Enzyme INP-clos EPcut | 30 |
3 | 2/19 Restriction Enzyme E1010 EPcut | 30 |
4 | blank | - |
![](https://static.igem.org/mediawiki/2016/6/6d/T--Kyoto--2_19_No18_Gel_Extraction_Notsu_10_15_08_27_59.1.jpg)
![](https://static.igem.org/mediawiki/2016/b/b6/T--Kyoto--2_19_No18_Gel_Extraction_Notsu_10_15_08_27_59.2.jpg)
2/19
PCR (Purification)
WanSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
2/19 Restriction Enzyme BAN-cex E, Pcut | 9.6 | 1.85 | 0.84 |
〃BAN-clos E, Pcut | 18.9 | 1.83 | 0.71 |
〃INP-cex E, Pcut | 14.9 | 1.77 | 1.46 |
〃INP-clos E, Pcut | 13.4 | 1.73 | 1.67 |
〃E1010 E, Pcut | 19.6 | 1.86 | 0.40 |
2/19
Transformation
Wan, Egashira, KiminsertDNA (µl) | VectoeDNA (µl) | ligation high (µl) | total (µl) |
---|---|---|---|
BAN-cex/4 | E1010(pSB1C3)/2 | 6 | 12 |
BAN-clos/3 | 〃/3 | 6 | 12 |
INP-cex/4.5 | 〃/1.5 | 6 | 12 |
INP-clos/4.5 | 〃/1.5 | 6 | 12 |
2/19
Transformation
WanSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/19 Ligation BAN-cex+E1010 | 10 | DH5a/(20) | LB(CP) |
〃BAN-clos+E1010 | 10 | DH5a/(20) | LB(CP) |
〃INP-cex+E1010 | 10 | DH5a/(20) | LB(CP) |
〃INP-clos+E1010 | 10 | DH5a/(20) | LB(CP) |
2/19
Restriction Enzyme Digestion
Michimori, KimSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/18 purification m.scFv EPcut+K592009 EPcut colony1 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony2 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony3 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony4 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/19
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 2/19 restricion enzyme digestion colony1 /10 | |
3 | 2/19 restricion enzyme digestion colony2 /10 | |
4 | 2/19 restricion enzyme digestion colony3 /10 | |
5 | 2/19 restricion enzyme digestion colony4 /10 |
2/19
Liquid Culture
Egashira, WanName | medium |
---|---|
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4 | LB(CP) |
2/22
Colony PCR (typeA)
YamadaTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/19 transformation BAN-His-CBDcex colony1 | vf 1.25 vr 1.25 | 10 | 12.5 | 25 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/19 transformation BAN-His-CBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/19 transformation INP-His-CBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/22
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | ladder 1kb/(5) | / |
2 | 2/22 PCR BAN-His-CBDcex colony1/(10) | 515 |
3 | 〃colony2/(10) | 515 |
4 | 〃colony3/(10) | 515 |
5 | 2/22 PCR BAN-His-CBDclos colony3/(10) | 487 |
6 | 2/22 PCR INP-His-CBDcex colony1/(10) | 405 |
7 | 〃colony2/(10) | 405 |
8 | 〃colony3/(10) | 405 |
9 |
![](https://static.igem.org/mediawiki/2016/e/e5/T--Kyoto--2_22_No23_Electrophoresis_Kosaka_09_24_12_30_17.jpg)
2/22
Liquid Culture
WanName | medium |
---|---|
2/22 PCR BAN-His-cex colony1 | CP/5ml |
〃colony2 | 〃 |
〃colony3 | 〃 |
2/22 PCR BAN-His-clos colony3 | 〃 |
2/22
Miniprep
Egashira, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony2 | 137.4 | 1.89 | 2.12 |
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony3 | 164.0 | 1.89 | 2.09 |
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony4 | 138.1 | 1.86 | 2.16 |
2/23
Ligation
Wan, YamamotoinsertDNA (µl) | VectoeDNA (µl) | ligation high (µl) | total (µl) |
---|---|---|---|
BAN-cex/4 | E1010cpsb1c3/2 | 6 | 12 |
BAN-clos/3 | 〃/3 | 6 | 12 |
INP-cex/4.5 | 〃/1.5 | 6 | 12 |
INP-clos/4.5 | 〃/1.5 | 6 | 12 |
2/23
Transformation
WanSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/23 Ligation BAN-cex+E1010 | 2 | DH5a/(20) | LB(CP) |
〃clos〃 | 2 | 〃 | 〃 |
〃INP-cex〃 | 2 | 〃 | 〃 |
〃INP-clos〃 | 2 | 〃 | 〃 |
2/24
Colony PCR (typeA)
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/23 transformation BAN-His-CBDclos colony1 | vf1.25 vr1.25 | 10 | 12.5 | 25 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
2/23 transformation INP-His-CBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
2/23 transformation INP-His-CBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
2/24
Miniprep
YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/22 liqid culture BAN-Hi-cex colony1 | 45.3 | 1.88 | 2.08 |
〃colony2 | 47.1 | 1.89 | 1.98 |
〃colony3 | 43.8 | 1.96 | 2.08 |
2/22 BAN-His-clos colony3 | 53.7 | 1.82 | 1.62 |
2/24
Liquid Culture
EgashiraName | medium |
---|---|
2/23 transformation BAN-clos colony1 | LB(CP/5ml) |
〃colony2 | 〃 |
〃colony3 | 〃 |
2/23 transformation INP-cex colony2 | 〃 |
〃colony3 | 〃 |
2/23 transformation INP-clos colony1 | 〃 |
2/25
Miniprep
Egashira, KimDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/23 transformation BAN-His-CBDclos colony1 | 79.9 | 1.89 | 2.15 |
2/23 transformation BAN-His-CBDclos colony2 | 68.0 | 1.88 | 2.02 |
2/23 transformation BAN-His-CBDclos colony3 | 80.7 | 1.94 | 2.18 |
2/23 transformation INP-His-CBDcex colony2 | 106.0 | 1.91 | 2.17 |
2/23 transformation INP-His-CBDcex colony3 | 136.7 | 1.93 | 2.23 |
2/23 transformation INP-His-CBDclos colony1 | 183.7 | 1.91 | 2.21 |
2/25
Sequence
MichimoriSample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
---|---|---|---|---|
Bcx 1-F | 2/24 miniprep BAN-His-CBDcex colony1 /6.6 | VF /0.2 | 3.2 | 10 |
Bcx 1-R | 2/24 miniprep BAN-His-CBDcex colony1 /6.6 | VR /0.2 | 3.2 | 10 |
Bcx 2-F | 2/24 miniprep BAN-His-CBDcex colony2 /6.4 | VF /0.2 | 3.4 | 10 |
Bcx 2-R | 2/24 miniprep BAN-His-CBDcex colony2 /6.4 | VR /0.2 | 3.4 | 10 |
Bcl1-F | 2/25 miniprep BAN-His-CBDclos colony1 /3.8 | VF /0.2 | 6.0 | 10 |
Bcl1-R | 2/25 miniprep BAN-His-CBDclos colony1 /3.8 | VR /0.2 | 6.0 | 10 |
Bcl2-F | 2/25 miniprep BAN-His-CBDclos colony3 /3.7 | VF /0.2 | 6.1 | 10 |
Bcl2-R | 2/25 miniprep BAN-His-CBDclos colony3 /3.7 | VR /0.2 | 6.1 | 10 |
Icx1-F | 2/25 miniprep INP-His-CBDcex colony2 /2.8 | VF /0.2 | 7.0 | 10 |
Icx1-R | 2/25 miniprep INP-His-CBDcex colony2 /2.8 | VR /0.2 | 7.0 | 10 |
Icx2-F | 2/25 miniprep INP-His-CBDcex colony3 /2.2 | VF /0.2 | 7.6 | 10 |
Icx2-R | 2/25 miniprep INP-His-CBDcex colony3 /2.2 | VR /0.2 | 7.6 | 10 |
Icl1-F | 2/25 miniprep INP-His-CBDclos colony1 /1.6 | VF /0.2 | 8.2 | 10 |
Icl1-R | 2/25 miniprep INP-His-CBDclos colony1 /1.6 | VR /0.2 | 8.2 | 10 |
2/25
Liquid Culture
Egashira, YamadaName | medium |
---|---|
2/19 transformation BAN-His-cex colony1 | LB(CP/5ml) |
2/19 transformation BAN-His-cex colony2 | LB(CP/5ml) |
2/23 transformation BAN-His-clos colony1 | LB(CP/5ml) |
2/23 transformation BAN-His-clos colony3 | LB(CP/5ml) |
2/23 transformaation INP-His-cex colony2 | LB(CP/5ml) |
2/23 transformation INP-His-cex colony3 | LB(CP/5ml) |
INP-His-clos colony1 | LB(CP/5ml) |
2/25
Sequence
MichimoriSample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
---|---|---|---|---|
S2-F | 2/22 miniprep scFv colony2 /2.2 | VF /0.2 | 7.6 | 10 |
S2-R | 2/22 miniprep scFv colony2 /2.2 | VR /0.2 | 7.6 | 10 |
S3-F | 2/22 miniprep scFv colony3 /1.8 | VF /0.2 | 8.0 | 10 |
S3-R | 2/22 miniprep scFv colony3 /1.8 | VR /0.2 | 8.0 | 10 |
S4-F | 2/22 miniprep scFv colony4 /2.2 | VF /0.2 | 7.6 | 10 |
S4-R | 2/22 miniprep scFv colony4 /2.2 | VR /0.2 | 7.6 | 10 |
2/25
Liquid Culture
Egashira, Kim, YamadaName | medium |
---|---|
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4 | LB(CP) |
2/26
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
J23100 | VF/ 0.75 | J23100+RBSRv | 11 | PrimeSTARMax Premix/ 12.5 | 25 | |
J23108 | VF/ 0.75 | J23108+RBSRv | 11 | PrimeSTARMax Premix/ 12.5 | 25 | |
J23114 | VF/ 0.75 | J23114+RBSRv | 11 | PrimeSTARMax Premix/ 12.5 | 25 |
2/26
PCR (Target)
LiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
J23100 iGEM Part | VF /0.75 | J23100+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
J23108 iGEM Part | VF /0.75 | J23108+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
J23114 iGEM Part | VF /0.75 | J23114+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
2/26
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | - |
2 | 2/26 PCR J23100 /5 | 193+α |
3 | 2/26 PCR J23108 /5 | 193+α |
4 | 2/26 PCR J23114 /5 | 193+α |
2/26
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
J23100 iGEM Part /0.1 | VF 0.75 | J23100+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
J23108 iGEM Part /0.1 | VF 0.75 | J23108+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
J23114 iGEM Part /0.1 | VF 0.75 | J23114+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
2/26
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 2/26 PCR J23100 | 25 |
3 | 2/26 PCR J23108 | 25 |
4 | 2/26 PCR J23114 | 25 |
2/29
Gel Extraction(Measurement)
Egashira, WanSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
2/26 PCR J23100 | 1.5 | 2.71 | 0.09 |
2/26 PCR J23108 | 3.6 | 18.64 | 0.01 |
2/26 PCR J23114 | 1.7 | 3.05 | 0.13 |
2/29
PCR (Target)
Wan, KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
J23100 iGEM Part /0.2 | VF /1.5 | J23100+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
J23108 iGEM Part /0.2 | VF /1.5 | J23108+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
J23114 iGEM Part /0.2 | VF /1.5 | J23114+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
3/1
Electrophoresis
Li, KimLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 100bp ladder /6 | |
2,3 | J23114 /50 | 193+α |
4,5 | J23100 /50 | 193+α |
6,7 | J23108 /50 | 193+α |
3/1
PCR (Purification)
Yamamoto, KimSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
2/29 PCR J23114 | 4.7 | 1.59 | 0.01 |
2/29 PCR J23100 | 2.4 | 2.60 | 0.01 |
2/29 PCR J23108 | 3.8 | 4.51 | 0.01 |
3/1
PCR (Target)
YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/1 PCR purification J23100 /5 | VF /0.75 | J23100Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
3/1 PCR purification J23108 /5 | VF /0.75 | J23108Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
3/1 PCR purification J23114 /5 | VF /0.75 | J23114Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
2/22 miniprep m.scFv colony3 /0.5 | BAN+scFv /0.75 | VR /0.75 | - | 10.5 | Prime Star Premix /12.5 | 25 |
3/1
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | - |
2 | 3/1 PCR J23100 /5 | about 200 |
3 | 3/1 PCR J23108 /5 | about 200 |
4 | 3/1 PCR J23114 /5 | about 200 |
5 | 3/1 PCR m.scFv colony3 /5 | 949 |
3/1
Electrophoresis
Kim, YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 100bp ladder /5 | - |
2 | 3/1 OE-PCR J23100 + m.scFv colony3 /2 | about 1200 |
3 | 3/1 OE-PCR J23108 + m.scFv colony3 /2 | about 1200 |
4 | 3/1 OE-PCR J23114 + m.scFv colony3 /2 | about 1200 |
3/1
PCR (Purification)
Kim, YamadaSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/1 OE-PCR J23100 + m.scFv colony3 | 180.5 | 1.89 | 1.83 |
3/1 OE-PCR J23108 + m.scFv colony3 | 183.3 | 1.87 | 1.96 |
3/1 OE-PCR J23114 + m.scFv colony3 | 178.8 | 1.87 | 2.08 |
3/1
PCR (Target)
Kim, YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/1 PCR J23100 /1 + 3/1 PCR m.scFv colony3 /0.5 | VF /0.5 | VR /0.75 | - | 9.5 | Prime Star Premix /12.5 | 25 |
3/1 PCR J23108 /1 + 3/1 PCR m.scFv colony3 /0.5 | VF /0.5 | VR /0.75 | - | 9.5 | Prime Star Premix /12.5 | 25 |
3/1 PCR J23114 /2 + 3/1 PCR J23114 /0.5 | VF /0.5 | VR /0.75 | - | 8.5 | Prime Star Premix /12.5 | 25 |
3/1
Gel Extraction
Kim, YamadaLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | |
2 | 3/1 EPcut J23114 + m.scFv | 20 |
3 | 3/1 EPcut J23108 + m.scFv | 20 |
4 | 3/1 EPcut J23100 + m.scFv | 20 |
3/1
Restriction Enzyme Digestion
Li, Kim, YamadaSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/1 OE-PCR J23100 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/1 OE-PCR J23108 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/1 OE-PCR J23114 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/2
PartsAwaking
Michimori, Kim, YamadaDescription | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
---|---|---|---|---|---|---|
f1 origin | K314110 | pSB1C3 | 1A | 2016 | 1 | 10 |
T1 terminator from E. coli rrnB | K731722 | pSB1C3 | 1B | 2016 | 1 | 10 |
Promoter of the CaiF protein | K398326 | pSB1C3 | 1C | 2016 | 1 | 10 |
3/2
Transformation
MichimoriSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
3/2 Parts Awakening K314110 | 10 | DH5α /20 | LB(CP) |
3/2 Parts Awakening K731722 | 10 | DH5α /20 | LB(CP) |
3/2 Parts Awakening K398326 | 10 | DH5α /20 | LB(CP) |
3/2
Gel Extraction(Measurement)
YamadaSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
J23100+m.scFv 3/1 EPcut | 17.7 | 1.90 | 1.68 |
J23108+m.scFv 3/1 EPcut | 11.9 | 2.04 | 0.26 |
J23114+m.scFv 3/1 EPcut | 16.8 | 1.97 | 1.79 |
3/2
Restriction Enzyme Digestion
WanSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
E0020 | 40 | 2 | 0 | 0 | 2 | 10×H /6 | - | 10 | 60 |
3/2
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
J23100+pSB1C3 | 2 | DH5α /20 | LB(CP) |
J23108+pSB1C3 | 2 | DH5α /20 | LB(CP) |
J23114+pSB1C3 | 2 | DH5α /20 | LB(CP) |
3/3
PCR (Target)
YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/9 miniprep E1010 /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/24 miniprep BAN-His-CBDcex colony2 /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep BAN-His-CBDclos /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep INP-His-CBDcex /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep INP-His-CBDclos /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
3/3
Gel Extraction(Measurement)
EgashiraSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
3/2 EPcut E0020 | 23.1 | 1.85 | 1.69 |
3/3
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | |
2 | 3/2 EPcut E0020 (pSB1C3) | 20 |
3/3
Ligation
WanVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23100+m.scFv EPcut /12 | 16 | 32 |
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23108+m.scFv EPcut /12 | 20 | 40 |
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23114+m.scFv EPcut /12 | 16 | 32 |
3/3
Transformation
Wan, KimSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
J23100+PSB1C3 | 2 | DH5alpha/ 20 | LB(CP) |
J23108+PSB1C3 | 2 | DH5alpha/ 20 | LB(CP) |
J23114+PSB1C3 | 2 | DH5alpha | LB(CP) |
3/3
Liquid Culture
Wan, KimName | medium |
---|---|
K314110 colony1 | LB(CP) |
K731722 colony1 | LB(CP) |
K398326 colony1 | LB(CP) |
3/4
Transformation
Li, NotsuSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
J23100+pSB1C3 | 10 | DH5α /20 | LB(CP) |
J23108+pSB1C3 | 10 | DH5α /20 | LB(CP) |
J23114+pSB1C3 | 10 | DH5α /20 | LB(CP) |
3/7
Miniprep
YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
2/25 liquid culture BAN-His-CBDcex colony2 | 71.4 | 1.83 | 1.43 |
2/25 liquid culture BAN-His-CBDclos colony1 | 61.6 | 1.83 | 1.90 |
2/25 liquid culture INP-His-CBDcex colony3 | 83.4 | 1.94 | 1.92 |
2/25 liquid culture INP-His-CBDclos colony1 | 100.7 | 1.87 | 1.44 |
3/7
Transformation
WanSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
3/7 miniprep BAN-His-CBDcex | 2 | DH5α /20 | LB(CP) |
3/7 miniprep BAN-His-CBDclos | 2 | DH5α /20 | LB(CP) |
3/7 miniprep INP-His-CBDcex | 2 | DH5α /20 | LB(CP) |
3/7 miniprep INP-His-CBDclos | 2 | DH5α /20 | LB(CP) |
3/7
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
3/3 ligation J23100+pSB1C3 | DH5α /20 | LB(CP) | |
3/3 ligation J23108+pSB1C3 | DH5α /20 | LB(CP) | |
3/3 ligation J23114+pSB1C3 | DH5α /20 | LB(CP) |
3/7
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
3/4 transformtion J23100 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23100 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23108 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23108 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23114 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23114 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/7
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 2min | 1 |
Denature | 95℃ | 15sec | 35 |
Annealing | 55℃ | 15sec | 35 |
Extension | 72℃ | 5sec | 35 |
Final Elongation | 72℃ | 10min | 1 |
Final Holding | 4℃ | - | - |
3/7
Electrophoresis
Wan, YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 3/7 colony PCR J23100 colony1 /10 | 1000+α |
3 | 3/7 colony PCR J23100 colony2 /10 | 1000+α |
4 | 3/7 colony PCR J23108 colony1 /10 | 1000+α |
5 | 3/7 colony PCR J23108 colony2 /10 | 1000+α |
6 | 3/7 colony PCR J23114 colony1 /10 | 1000+α |
7 | 3/7 colony PCR J23114 colony2 /10 | 1000+α |
![](https://static.igem.org/mediawiki/2016/c/c9/T--Kyoto--3_7_No154_Electrophoresis_Notsu_10_02_10_08_42.jpg)
3/7
PCR (Steps)
YamadaSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Extension | 72℃ | 5min | 35 |
Final Elongation | 4℃ | - | - |
Final Holding | 4℃ | - | - |
3/7
PCR (Target)
YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/1 purification J23100 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23100 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23108 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23108 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23114 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23114 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/8
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
3/7 transformation BAN-CBDcex colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-CBDclos colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
3/8
PCR (Purification)
UchinoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/7 PCR J23100 | 423.3 | 1.84 | 2.20 |
3/7 PCR J23108 | 439.1 | 1.85 | 2.11 |
3/7 PCR J23114 | 451.2 | 1.81 | 1.91 |
3/8
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/8 purification J23100 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23108 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23114 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8
Gel Extraction(Measurement)
Li, Kim, YamadaSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
J23114 3/8 EPcut | 13.7 | 3.62 | 0.03 |
J23108 3/8 EPcut | 15.4 | 1.83 | 1.14 |
J23100 3/8 EPcut | 14.5 | 1.79 | 1.03 |
3/9
Miniprep
KimDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
K731722 | 67.2 | 1.70 | 1.02 |
K314110 | 28.9 | 1.86 | 1.14 |
K398326 | 95.6 | 1.64 | 0.94 |
3/9
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/1 miniprep K314110 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/1 miniprep K731722 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/1 miniprep K398326 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/9
Restriction Enzyme Digestion
LiSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/8 purification J23100 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23108 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23114 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23100 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
3/8 purification J23108 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
3/8 purification J23114 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
3/11
Gel Extraction
YamadaLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 3/10 E.P. cut J23100+scFv | 15 |
3 | 3/10 E.P. cut J23100+scFv | 15 |
4 | 3/10 E.P.cut J23108+scFv | 15 |
5 | 3/10 E.P.cut J23108+scFv | 15 |
6 | 3/10 E.P.cut J23114+scFv | 15 |
7 | 3/10 E.P.cut J23114+scFv | 15 |
8 | 3/9 E.P. cut J23100+scFv | 15 |
9 | 3/9 E.P. cut J23100+scFv | 15 |
10 | 3/9 E.P. cut J23119 scFv | 15 |
11 | 3/9 E.P. cut J23119 scFv | 15 |
12 | 3/9 E.P.cut J23114+scFv | 15 |
3/11
Gel Extraction
YamadaLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kbp ladder | 15 |
2 | 3/9 E.P. cut K314110 | 15 |
3 | 3/9 E.P. cut K314110 | 15 |
4 | 3/9 E.P cut K731722 | 15 |
5 | 3/9 E.P cut K731722 | 15 |
6 | 3/9 E.P cut K7398326 | 15 |
7 | 3/9 E.P cut K7398326 | 15 |
8 | 3/9 E.P> cut J23114+scFv | 15 |
![](https://static.igem.org/mediawiki/2016/9/9a/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C2.jpg)
![](https://static.igem.org/mediawiki/2016/8/8c/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C3.jpg)
![](https://static.igem.org/mediawiki/2016/c/ce/T--Kyoto--3_11_No164_Gel_Extraction_Yoshimoto_09_16_09_36_49%2C4.jpg)
3/11
PCR (Purification)
YoshimotoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/11 Gel Ex J23108+scFv | 18.9 | 1.90 | 0.19 |
3/11 Gel Ex J23108+scFv | 7.0 | 1.92 | 0.57 |
3/11 Gel Ex J23114+scFv | 5.3 | 2.37 | 0.25 |
3/11 Gel Ex J23100+scFv | 39.2 | 1.93 | 1.29 |
3/11 Gel Ex J23108+scFv | 33.6 | 1.82 | 0.68 |
3/11 Gel Ex J23114+scFv | 27.8 | 1.86 | 1.25 |
3/11 Gel Ex pSCIC3 | 29.0 | 1.86 | 1.42 |
3/11
Ligation
KimVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23100+scfv/ 5.6 | 10.6 | 21.2 |
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23108+scfv/ 5 | 11.5 | 23 |
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23114+scfv/ 5 | 12.8 | 25.6 |
3/11
Transformation
KimSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
3/11 Ligation PSB1C3+J23100+scFV | DH5α /20 | LB | |
3/11 Ligation PSB1C3+J23108+scFv | DH5α /20 | LB | |
3/11 Ligation PSB1C3+J23114+scFv | DH5α /20 | LB |
3/14
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kbp ladder | |
2 | 3/8 PCR BANcex colony1 | 515 |
3 | 3/8 PCR BAN-cex colony2 | 515 |
4 | 3/8 PCR BAN-clos colony1 | 487 |
5 | 3/8 PCR BAN-clos-colony2 | 487 |
6 | 3/8 PCR INP-cex colony 1 | 1405 |
7 | 3/8 PCR INP-cex colony 2 | 1405 |
8 | 3/8 PCR INP-clos colony 1 | 1378 |
9 | 3/8 PCR INP-clos colony 2 | 1378 |
![](https://static.igem.org/mediawiki/2016/a/a6/T--Kyoto--3_14_No43_Electrophoresis_Yoshimoto_09_02_04_10_33.jpg)
3/14
PCR (Target)
Uchino, EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/25 INPcex colony2 | VF 0.75 CBDcex ctl. Rv0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/25 INPcex colony2 | CBDcex ctl Fw 0.75 VR0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/18 INPclos Gel Extraction | VF 0.75 CBD clos ctl. Rv0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl Fw 0.75 VR0.75 | 11 | KAPA HiFi 12.5 | 25 |
3/14
Gel Extraction
Uchino, EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
4 | 3/14PCR INP cex (VF CBD cex ctl.Rv)25 | 5 |
5 | 3/14PCR INPcex (CBD cex ctl.Fw VR) 25 | 5 |
6 | 3/14PCR INP clos(VF CBDclos ctl.Rv)25 | 5 |
7 | 3/14PCR INPclos (CBDclos ctl.Fw VR) 25 | 5 |
![](https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--3_14_No45_Gel_Extraction_Yoshimoto_09_02_09_48_51.1.jpg)
![](https://static.igem.org/mediawiki/2016/5/56/T--Kyoto--3_14_No45_Gel_Extraction_Yoshimoto_09_02_09_48_51.2.jpg)
3/14
Restriction Enzyme Digestion
NotsuSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/7 miniprep BAN-His-CBDcex colony2 | 30 | - | - | 1 | 1 | 4 | - | 4 | 40 |
3/7miniprep BAN-His-CBDclos colony1 | 30 | - | - | 1 | 1 | 4 | - | 4 | 40 |
3/7miniprep INP-His-CBDcex colony3 | 30 | - | - | 1 | 1 | 4 | - | 4 | 40 |
3/'miniprep INP-His-CBDcloscolony 1 | 40 | - | - | 1 | 1 | 5 | - | 3 | 50 |
2/9 miniprep J23105 | 61 | - | 1 | - | - | - | - | 0 | 70 |
3/14
OE-PCR (typeA)
KimTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
3/14 Gel Ex INP cex 0.5 VR ct Fw + 3/14 Gel Ex INP cex 0.5 VF ct Rv | VF1.5 VR1.5 | 21 | 25 | 50 |
3/14 Gel Ex INP clos 0.5 VR ct Fw + 3/14 Gel Ex INP clos 0.5 VF ct Rv | VF1.5 VR1.5 | 21 | 25 | 50 |
3/14
PCR (Steps)
KimSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 95℃ | 5min | 1 |
Denature | 78℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Extension | 72℃ | 45sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
3/14
Colony PCR (typeA)
UchinoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/8 INP His9 | VF 0.75 scFv ctl. Rv 0.75 | 11 | 12.5 | 25 |
2/8 INP His9 | scFv ctl Fw 0.75 VR 0.75 | 11 | 12.5 | 25 |
2/25 INPcex colony2 | VF 0.75 CBDcex ctl. Rv0.75 | 11 | 12.5 | 25 |
2/25 INPcex colony2 | CBDcex ctl. Fw 0.75 VR 0.75 | 11 | 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl. Rv 0.75 VF 0.75 | 11 | 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl Fw 0.75 | 11 | 12.5 | 25 |
3/14
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 3/14 PCR INP His9 (VF scFv.ctl. Rv) | 25 |
3 | 3/14 PCR INP His9 (scFv ctl.Fw VR) | 25 |
![](https://static.igem.org/mediawiki/2016/1/11/T--Kyoto--3_14_No173_Gel_Extraction_Yoshimoto_09_16_10_54_56%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/c/ce/T--Kyoto--3_14_No173_Gel_Extraction_Yoshimoto_09_16_10_54_56%2C2.jpg)
3/15
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder 5 | |
2 | 3/14 OEPCR CBDcex ctl 50 | 10 |
3 | 3/14 OEPCR CBDclos ctl 50 | 10 |
3/15
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/25 INPcex colony2 | VF /1.5 | CBDcex.ctl.RV /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPcex colony2 | CBDcex.ctl.Fw /1.5 | VR /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPclos colony1 | VF /1.5 | CBDclos.ctl.Rv /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPclos colony1 | CBDclos.ctl.Fw /1.5 | VR /1.5 | - | 22 | KAPA HiFi /25 | 50 |
3/15
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
4 | 3/15 PCR INPcex(VF 20, CBDcex ctl.Rv 20) | 4 |
5 | 3/15 PCR INPcex(CBDcex ctl.Fw20, VR 20) | 4 |
6 | 3/15 PCR INPclos(VF 20 CBDclos ctl.Rv 20) | 4 |
7 | 3/15 PCR INPclos(CBDclos ctl.Fw20, VR 20) | 4 |
3/15
Liquid Culture
Li, UchinoName | medium |
---|---|
3/11 scFv J23100 colony1,4 colony2,3(plate2) | LB(CP) 5ml×2 |
3/11 scFv J23108 colony 2(plate1) colony(plate2) | LB(CP) 5ml×2 |
3/14 scFv J23114 colony 1,2,4(plate1) colony1,2,(plate2) | LB(CP) 5ml×2 |
3/16
PCR (Purification)
Kim, YamadaSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/15 Gelex CBDcex ctl. Fw VR | 55.3 | 1.95 | 0.11 |
3/15 Gelex CBDcex VF ctl.RV | 8.2 | 1.74 | 1.04 |
3/15Gelex CBDclos ctl Fw VR | 41.2 | 1.85 | 1.51 |
3/15Gelex CBDclos VF ctl RV | 10.6 | 1.56 | 0.60 |
3/15Gelex BANcexSP | 34.9 | 1.70 | 1.84 |
3/15Gelex BANclosSP | 32.0 | 1.95 | 0.07 |
3/15 Gelex INPcex SP | 40.3 | 1.76 | 1.76 |
3/15Gelex INPclosSP | 106.8 | 1.83 | 0.19 |
3/16
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
3 | 3/15 PCR INPcex(VF 20, CBDcex ctl, RV 20) | 4 |
4 | 3/15 PCR INPclos(VF 20, CBDclos ctl, RV 20) |
3/16
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
4 | CBDcex ctl | |
5 | CBDclos ctl |
3/16
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/15 Gel Ex INPcex 0.5(VF ctl.RV) 3/15 Gel Ex INPcex0.2(ctl.Fw VR) | VF1.25 | VR1.25 | KAPA taq 12.5 | 9.3 | 25 | |
3/15 GEl EX INPclos 0.5(VF ctl RV) 3/15Gel Ex INPclos0.2(ctl.Fw VR) | VF1.25 | VR1.25 | KAPA taq 12.5 | 9.3 | 25 |
3/16
PCR (Target)
KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/14 Gel Extraction INP-His9 VR/ct/Rv /0.5 + 3/14 Gel Extraction INP-His9 /0.5 | VF /1.5 | VR /1.5 | - | 21 | 2×KAPA HiFi /25 | 50 |
3/16
PCR
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | KAPA HiFi/(µl) | Total/(µl) |
---|---|---|---|---|
2/8 INP HIs9 | VF 1.5 scFvctl. RV1.5 | 22 | 25 | 50 |
2/8 INP His9 | VR1.5 scFvctl. Fw 1.5 VR1.5 | 22 | 25 | 50 |
3/16
Miniprep
Egashira, YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
J23100 scFv c1-p1 | 135.7 | 1.85 | 1.76 |
J23100 scFv C4-p1 | 135.1 | 1.86 | 1.85 |
J23100 scFv c3-p2 | 155.0 | 1.87 | 2.04 |
J23100 scFv c3-p2 | 205.9 | 1.76 | 1.32 |
J23108 scFv c2-p1 | 166.5 | 1.88 | 2.06 |
J23108 scFvc1-p2 | 95.7 | 1.84 | 1.83 |
J23114 scFv c1-p1 | 145.7 | 1.80 | 1.99 |
J23114 scFv c2-p1 | 111.7 | 1.90 | 2.16 |
J23114 scFv c4-p1 | 169.2 | 1.90 | 2.11 |
J23114 scFv c1-p2 | 127.4 | 1.87 | 2.00 |
J23114 scFv c2-p2 | 136.2 | 1.90 | 2.13 |
3/16
Sequence
MichimoriSample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
---|---|---|---|---|
100A-F | 3/15 miniprep J23100 scFv c1-p1 /2.2 | VF /0.2 | 7.6 | 10 |
100A-R | 3/15 miniprep J23100 scFv c1-p1 /2.2 | VR /0.2 | 7.6 | 10 |
100B-F | 3/15 miniprep J23100 scFv c4-p1 /2.2 | VF /0.2 | 7.6 | 10 |
100b-R | 3/15 miniprep J23100 scFv c4-p1 /2.2 | VR /0.2 | 7.6 | 10 |
100C-F | 3/15 miniprep J23100 scFv c2-p2 /1.9 | VF /0.2 | 7.9 | 10 |
100C-R | 3/15 miniprep J23100 scFv c2-p2 /1.9 | VR /0.2 | 7.9 | 10 |
100D-F | 3/15 miniprep J23100 scFv c3-p2 /1.5 | VF /0.2 | 8.3 | 10 |
100D-R | 3/15 miniprep J23100 scFv c3-p2 /1.5 | VR /0.2 | 8.3 | 10 |
108A-F | 3/15 miniprep J23108 scFv c2-p1 /1.8 | VF /0.2 | 8.0 | 10 |
108A-R | 3/15 miniprep J23108 scFv c2-p1 /1.8 | VR /0.2 | 8.0 | 10 |
108B-F | 3/15 miniprep J23108 scFv c1-p2 /3.1 | VF /0.2 | 6.7 | 10 |
108B-R | 3/15 miniprep J23108 scFv c1-p2 /3.1 | VR /0.2 | 6.7 | 10 |
114A-F | 3/15 miniprep J23114 scFv c1-p1 /2.1 | VF /0.2 | 7.7 | 10 |
114A-R | 3/15 miniprep J23114 scFv c1-p1 /2.1 | VR /0.2 | 7.7 | 10 |
114B-F | 3/15 miniprep J23114 scFv c2-p1 /2.7 | VF /0.2 | 7.1 | 10 |
114B-R | 3/15 miniprep J23114 scFv c2-p1 /2.7 | VR /0.2 | 7.1 | 10 |
3/16
Sequence
MichimoriSample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
---|---|---|---|---|
114C-F | 3/15 miniprep J23114 scFv c4-p1 /1.8 | VF /0.2 | 8.0 | 10 |
114C-R | 3/15 miniprep J23114 scFv c4-p1 /1.8 | VR /0.2 | 8.0 | 10 |
114C-F | 3/15 miniprep J23114 scFv c1-p2 /2.4 | VF /0.2 | 7.4 | 10 |
114C-R | 3/15 miniprep J23114 scFv c1-p2 /2.4 | VR /0.2 | 7.4 | 10 |
114C-F | 3/15 miniprep J23114 scFv c2-p2 /2.2 | VF /0.2 | 7.6 | 10 |
114C-R | 3/15 miniprep J23114 scFv c2-p2 /2.2 | VR /0.2 | 7.6 | 10 |
3/16
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
2 | 3/15 PCR INP His9(VF scFv CH. Rv) | 20 |
3 | 3/15 PCR INP His9 (scFv ctl, Fw VR) | 20 |
3/16
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 3/15 PCR INP His9 (VF scFv ctl RV) | 20 |
3/16
Gel Extraction
EgashiraLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | |
2 | scFv (VF ctl.Rv) | |
3 | scFv ctl |
3/16
OEPCR (Target)
EgashiraSample1/(µl) | Sample2/(µl) | Primer/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/15 Gel Extraction scfv (VF ctl.Rv)/ 0.5 | 3/15 Gel Extraction scfv (ctl.Fw VR) | VF/ 1.25, VR/1.25 | - | 9.3 | KAPA Taq/ 12.5 | 25 |
3/16
PCR
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
scFv (VF ctl.RV) 0.5 | VF 1.25 ctl.Rv) 1.25 | 9.5 | 12.5 | 25 |
3/16
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.25+3/16 Gel Extraction scFv(ctl.Fw.VR) /0.25 | VF /1.25 | VR /1.25 | - | 9.5 | 2×KAPA HiFi /12.5 | 25 |
3/17
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/16 Gel Ex scFv(VF ctl.Rv)0.25, 3/15 Gel EX CBDcex(ctl.Fw VR) 0.25 | VF 1.25 | VR1.25 | KAPA Taq 12.5 | 9.5 | 25 | |
3/16 Gel Ex scFv(VF ctl.Rv) 0.25, Gel Ex CBDclos(ctl,Fw VR) 0.25 | VF 1.25 | VR1.25 | KAPA Taq 12.5 | 9.5 | 25 |
3/17
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
3 | 3/16 OEPCR CBDcex ctl 25 | 500 |
4 | 3/16 OEPCR CBDclos ctl 25 | 500 |
![](https://static.igem.org/mediawiki/2016/8/84/T--Kyoto--3_17_No59_Electrophoresis_Yoshimoto_09_03_12_05_46.jpg)
3/17
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
2 | CBDcex ctl 25 | |
3 | CBDclos ctl 25 | |
4 | 1kb ladder 5 |
3/17
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/16 Gel Ex scFv(VF ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 | VF 1.25 | VR1.25 | KAPA 12.5 | 9.8 | 25 | |
3/16 Gel Ex scFv(VF ctl,Rv) 0.1, 3/15 GelEx CBDclos(ctl,Fw VR) 0.1 ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 | VF 1.25 | VR1.25 | KAPA 12.5 | 9.8 | 25 |
3/17
Miniprep
YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
3/8 Liquid Culture BAN-His-cex | 39.3 | 1.91 | 1.67 |
3/8 Liquid Culture BAN-His=clos | 45.1 | 1.84 | 1.44 |
3/17
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 95℃ | 3min | 1 |
Denature | 95℃ | 15sec | 40 |
Annealing | 55℃ | 15sec | 40 |
Elongation | 72℃ | 1min | 40 |
Final Elongation | 72℃ | 1min | 1 |
Final Holding | 4℃ | - | - |
3/17
PCR (Target)
YamamotoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/17 miniprep BAN-His-cex | CBDcexFw 1.25 | VR1.25 | 12.5 | 9 | 25 | |
3/17 miniprep BAN-His-clos | CBDclosFw 1.25 | VR1.25 | 12.5 | 9 | 25 | |
2/9 miniprep J23105_1 | VF 1.25 | RFP-cex RV 1.25 | 12.5 | 9 | 25 | |
2/9 miniprep J23105_1 | VF 1.25 | RFP-clos Rv 1.25 | 12.5 | 9 | 25 |
3/17
PCR (Target)
EgashiraTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.1+3/15 Gel Extraction scFv(ctl.Fw.VR) /0.1 | VF /1.25 | VR /1.25 | - | 9.8 | 2×KAPA HiFi /12.5 | 25 |
3/18
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 3/10 PCR BAN-cex CBDcexFW VR | 25 |
3 | 3/10 PCR BAN-clos CBDclosFw VR | 25 |
4 | 3/10 PCR J23105 VF RFPcexRv | 25 |
5 | 3/10 PCRJ23105 VF RFPclos | 25 |
![](https://static.igem.org/mediawiki/2016/6/62/T--Kyoto--3_18_No63_Gel_Extraction_Yoshimoto_09_03_12_29_48.1.jpg)
![](https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--3_18_No63_Gel_Extraction_Yoshimoto_09_03_12_29_48.2.jpg)
3/18
Gel Extraction(Measurement)
YamamotoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
3/17 PCR BAN-CBDcex CBDcex.Fw+Rv | 127.7 | 1.90 | 1.57 |
3/17 PCR BAN-CBDclos CBDclos.Fw+VR | 93.4 | 1.92 | 2.19 |
3/17 PCR J23105 VF+RFP-CBDcex.Rv | 104.0 | 1.84 | 1.50 |
3/17 PCR J23105 VF+RFP-CBDclos.Rv | 123.0 | 1.87 | 1.84 |
3/18
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 95℃ | 3min | 1 |
Denature | 95℃ | 15sec | 40 |
Annealing | 60℃ | 15sec | 40 |
Elongation | 72℃ | 1min | 40 |
Final Elongation | 72℃ | 1min | 1 |
Final Holding | 4℃ | - | - |
3/18
PCR (Target)
YamamotoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/18 Gel Extraction BAN-His-cex 0.1 cexFw+VR , 3/18Gel Extraction J23105 VF+RFP-cexRv | VF 1.25 | VR 1.25 | KAPATaq 12.5 | 9.8 | 25 | |
3/18 Gel Extraction BAN-His-clos 0.1 closFw+VR , 3/18Gel Extraction J23105 VF+RFP-closRv | VF 1.25 | VR 1.25 | KAPATaq 12.5 | 9.8 | 25 |
3/18
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | |
2 | 3/18 OEPCR RFP-cex | |
3 | 3/18 OEPCR RFP-clos |
![](https://static.igem.org/mediawiki/2016/6/69/T--Kyoto--3_18_No66_Gel_Extraction_Yoshimoto_09_04_05_26_17.jpg)
3/18
PCR (Target)
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/25 INP cex colony2 0.1×2 | VF 0.75×2 ctl RV 0.75 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | ctl Fw 0.75×2 VR 0.75×2 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | VF 0.75×2 VR 0.75×2 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | nothing | 12.4 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | VF ctl.Rv 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | ctl.Rv 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | VF 0.75×2 VR 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | nothing | 12.4 | 12.5×2 | 25×2 |
3/18
Colony PCR (typeA)
NotsuTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INP-His=CNDcex+RFP colony 1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDcex+RFP colony (3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDclos+RFP colony1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDclos+RFP colony 2(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDcex+RFP colony1( 3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDcex+RFP colony2 (3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDclos+RFP colony1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDclos+RFP colony2(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
3/18
Electrophoresis
Kim, YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder 5 | |
2 | 1 kb ladder 5 | |
3 | 2/25 INPcex colony2 10 | |
4 | 2/25 INPcex colony2 10 | |
5 | 2/25 INPcex colony2 10 | |
6 | 2/25 INPcex colony210 | |
7 | 2/25 INPclos colony1 10 | |
8 | 2/25 INPclos colony1 10 | |
9 | 2/25 INPclos colony1 10 | |
10 | 2/25 INPclos colony1 10 | |
11 | 100bp ladder 5 | |
12 | 1kbp ladder 5 |
![](https://static.igem.org/mediawiki/2016/8/88/T--Kyoto--3_18_No69_Electrophoresis_Yoshimoto_09_04_05_54_18.jpg)
3/18
PCR
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INP HIs9 0.1x2 | VF 0.75x2 ctl. Rv 0.75x2 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | ctl.Fw 1.75 VR 0.75 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | VF 0.75 VR 0.75 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | nothing | 12.4 | 12.5x2 | 25x2 |
3/22
Electrophoresis
NotsuLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /7 | |
2 | INP-His-CBDcex+RFP colony1 (3/19 PCR) /20 | |
3 | INP-His-CBDcex+RFP colony2 (3/19 PCR) /20 | |
4 | blank | |
5 | INP-His-CBDclos+RFP colony1 (3/19 PCR) /20 | |
6 | INP-His-CBDclos+RFP colony2 (3/19 PCR) /20 | |
7 | blank | |
8 | BAN-His-CBDcex+RFP colony1 (3/19 PCR) /20 | |
9 | BAN-His-CBDcex+RFP colony2 (3/19 PCR) /20 | |
10 | blank | |
11 | BAN-His-CBDclos+RFP colony1 (3/19 PCR) /20 | |
12 | BAN-His-CBDclos+RFP colony2 (3/19 PCR) /20 | |
13 | blank | |
14 | 100bp ladder /7 | |
15 | - | |
16 | - | |
17 | - |
![](https://static.igem.org/mediawiki/2016/7/70/T--Kyoto--3_22_No72_Electrophoresis_Notsu_10_16_12_57_19.jpg)
3/22
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
7 | 2/25 INPcexcolony2 (5) 10 | |
8 | 2/25 INPcexcolony2 (6) 10 | |
9 | 2/25 INPcexcolony2 (7) 10 | |
10 | 2/25 INPcexcolony2 (8) 10 | |
11 | 2/25 INPclos colony1(9) 10 | |
12 | 2/25 INPclos colony1(10) 10 | |
13 | 2/25 INPclos colony1(11) 10 | |
14 | 2/25 INPclos colony1(12) 10 | |
16 | 100b ladder 5 | |
17 | 1kb ladder 5 |
3/22
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
5 | 3/22 PCR CBDcex(VF/ctl.Rv) 10 | |
6 | 3/22 PCR CBDcex(ctl.Fw/VR) 10 | |
7 | 3/22 PCR CBDclos(VP/ctl.RV) 10 | |
8 | 3/22 PCR CBDclos(ctl.Fw/VR) 10 |
3/22
PCR (Target)
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
2/25 INPcex colony2 0.1 | VF 1.5 ctl, Rv 1.5 | 21.9 | 25 | 50 |
2/25 INPcex colony2 0.1 | ctl.Fw 1.5 ,VR 1.5 | 21.9 | 25 | 50 |
2/25 INPclos colony1 0.1 | VF 1.5, ctl,Rv 1.5 | 21.9 | 25 | 50 |
2/25 INPclos colony1 0.1 | ctl,Fw 1.5 VR 1.5 | 21.9 | 25 | 50 |
3/22
Miniprep
Notsu, YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
BANcex+RFP col.1 | 147.8 | 1.89 | 2.18 |
BANcex+RFP col.2 | 133.9 | 1.87 | 2.18 |
BANcex+RFP col.1 | 132.4 | 1.85 | 2.19 |
BANclos+RFP col.2 | 126.4 | 1.85 | 2.22 |
INPcex+RFP col.1 | 179.3 | 1.87 | 2.18 |
INPcex+RFP col.2 | 144.9 | 1.87 | 2.19 |
INPclos+RFPcol.1 | 152.2 | 1.87 | 2.23 |
INPclos+RFPcol.2 | 152.4 | 1.86 | 2.19 |
3/22
PCR (Target)
YamadaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 INP-His9 0.1 | VF 1.5 | ctl.Rv 1.5 | 25 | 21.9 | 50 | |
2/8 INP-His9 0.1 | scFv ctl.Fw 1.5 | VR 1.5 | 25 | 21.9 | 50 |
3/22
Miniprep
Egashira, KimDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
3/22 PCR scFv (VF/ctl.RN) | 105.4 | 1.81 | 1.54 |
3/22 PCR scFv (ctl. FV/VR) | 127.4 | 1.87 |
3/22
OEPCR (Target)
EgashiraSample1/(µl) | Sample2/(µl) | Primer/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/23 Gel Extraction scfv (VF ctl.Rv)/ 0.1 | 3/23 Gel Extraction scfv (ctl.Fw VR)/ 0.1 | VF/ 1.25, VR/1.25 | - | 10.8 | KAPA Taq/ 12.5 | 25 |
3/23
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
5 | 3/22 PCR CBDcex(VF/ctl.Rv) 40 | |
6 | 3/22 PCR CBDcex(ctl.Fw/VR) 40 | |
7 | 3/22 PCR CBDclos(VF/ctl.Rv) 40 | |
8 | 3/22 PCR CBDclos(ctl.Fw/VR) 40 |
3/23
PCR (Steps)
KimSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 95℃ | 2min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Elongation | 72℃ | 15sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 10℃ | - | - |
3/23
PCR (Target)
KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/18 Gelex BANcex Fw+VR 0.1, 3?18 Gelex J23105 VF+cexRv 0.1 | VF1.25 | VR 1.25 | 12.5 | 9.8 | 25 | |
3/18 Gelex BANclos closFV VR 0.1, 3/18Gelex J23105 VF+closR 0.1 | VF 1.25 | VR 1.25 | 12.5 | 9.8 | 25 |
3/23
Gel Extraction (Measurement)
EgashiraSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/22 PCR CBDcex(ctlFw/VR) | 26.9 | 1.75 | 1.04 |
3/22PCR CBDclos(ctl Fw/VR) | 100.6 | 1.90 | 1.65 |
3/23
Electrophoresis
KimLane | sample/(µl) | Length(bp) |
---|---|---|
3 | 3/18 OEPCR BANcex cexFw+VR J23105 VF+cexRv 25 | |
4 | BANclos clos Fw+VR J23105 VF+clos 25 | |
1 | 1kb ladder 10 | |
2 | 100bp ladder 10 |
![](https://static.igem.org/mediawiki/2016/1/1e/T--Kyoto--3_23_No78_Electrophoresis_Yoshimoto_09_04_06_47_22.jpg)
3/23
Electrophoresis
EgashiraLane | sample/(µl) | Length(bp) |
---|---|---|
4 | 3/23 OEPCR CBDcex ctl 25 | |
5 | 3/23 OEPCR CBDclos ctl 25 |
3/23
PCR (Target)
EgashiraTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
3/23 Gel Ex scFv(VF/ctl.Rv) 0.1, 3/23 Gel Ex CBDcex(ctl.Fw/VR) 0.1 | VF 0.75 VR 0.75 | 10.8 | 12.5 | 25 |
3/23 Gel Ex scFv (VF/ctl.Rv), 3/23 Gel Ex clos(ctl.Fw/VR) | VF 0.75 VR 0.75 | 10.8 | 12.5 | 25 |
3/23
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder 5 | |
2 | 100b ladder 7.5 | |
3 | 3/22 PCR scFv(VP/ctl.Rv) 40 | |
4 | 3/22 PCR scFv(ctl.Fw/VR) |
![](https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--3_23_No199_Electrophoresis_Koide_09_19_03_50_56%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/c/c4/T--Kyoto--3_23_No199_Electrophoresis_Koide_09_19_03_50_56%2C2.jpg)
3/24
PCR (Steps)
KimSteps | Temparature | Time | Cycle |
---|---|---|---|
Pre Denature | 95℃ | 2min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Elongation | 72℃ | 15sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 10℃ | - | - |
3/24
PCR (Target)
KimTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
3/18 Gelcex BAN cex cexFv VR 0.2, 3/18 Gelex J23105 VF cexFv 0.2 | VF1.25 | VR 1.25 | 12.5 | 9.6 | 25 | |
3/18 Gelcex BAN clos clos Fv VR 0.2, 3/18 Gelex J23105 VF clos Rv 0.2 | VF 1.25 | VR 1.25 | 12.5 | 9.6 | 25 |
3/24
PCR (Purification)
EgashiraSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/24 OEPCR CBDcex ctl | 468.6 | 1.82 | 2.06 |
3/24 OEPCR CBDclos ctl | 321.8 | 1.86 | 0.45 |
3/24
PCR (Purification)
KimDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
3/24 OEPCR BANcex ctl | 77.2 | 1.81 | 1.40 |
3/24 OEPCR BAN clos ctl | 23.8 | 1.75 | 1.56 |
3/24
PCR (Purification)
EgashiraSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
3/24 OEPCR scFv ctl | 394.2 | 1.83 | 1.19 |
3/24
Restriction Enzyme Digestion
EgashiraSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/24 OE PCR scFv ctl | 5.1 | 1 | 1 | 3 | 19.9 | 30 |
3/24
Transformation
EgashiraSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
PCBTC3+scFv+ctl. | DH5α | LB(CP) |
3/25
Restriction Enzyme Digestion
EgashiraSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/24 OEPCR CBDcex ctl | 4.3 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20.7 | 30 |
3/24 OEPCR CBDclos ctl | 6.2 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 18.8 | 30 |
3/15 J23100+scFv C3-P2 | 9.7 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15.3 | 30 |
3/25
Restriction Enzyme Digestion
KimSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
3/24 OEPCR BAN-CBDcex ctl | 10 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15 | 30 |
3/24 OEPCR BAN-CBDclos ctl | 10 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15 | 30 |
3/25
Ligation
YamadaVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
3/24 EPcut pSB1C3 /8 | 3/24 EPcut BAN-CBDcex.ctl /3.7 | 11.7 | 23.4 |
3/24 EPcut pSB1C3 /7 | 3/24 EPcut BAN-CBDclos.ctl /4.7 | 11.7 | 23.4 |
3/25
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
pSB1C3+BAN-CBDcex.ctl 3/25 ligation | - | 20 | LB(CP) |
pSB1C3+BAN-CBDclos.ctl 3/25 ligation | - | 20 | LB(CP) |
pSB1C3+CBDcex 3/24 ligation | - | 20 | LB(CP) |
pSB1C3+CBDclos 3/24 ligation | - | 20 | LB(CP) |
pSB1C3+scFv.ctl 3/24 ligation | - | 20 | LB(CP) |
3/28
Colony PCR (typeA)
YamadaTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
BANcex ctl col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BAncex ctl col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BANclos ctl col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BANclos ctl col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDcex col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDcex col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDclos col,1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDclos col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
scFv ctl col.1 | ||||
scFv ctl col.2 |
3/28
Electrophoresis
Lane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder(10) | |
2 | 100bb ladder(10) | |
3 | BANcex ctl col.1 | |
4 | BANcex ctl col.2 | |
5 | BANclos ctl col.1 | |
6 | BANclos ctl col.2 | |
7 | CBDcex col.1 | |
8 | CBDcex col.2 | |
9 | CBDclos col.1 | |
10 | CBDclos col.2 | |
11 | scFv ctl col.1 | |
12 | scFv ctl col.2 |
![](https://static.igem.org/mediawiki/2016/2/2d/T--Kyoto--3_28_No87_Electrophoresis_Sukegawa_09_19_03_19_19.jpg)
7/21
Liquid Culture
YamamotoName | medium |
---|---|
BclA-His-scFv glycerol stock1 | LB(CP) |
BclA-His-scFv glycerol stock2 | LB(CP) |
BclA-His-CBDcex glycerol stock1 | LB(CP) |
BclA-His-CBDcex glycerol stock2 | LB(CP) |
BclA-His-CBDclos glycerol stock1 | LB(CP) |
BclA-His-CBDclos glycerol stock2 | LB(CP) |
INP-His-CBDcex glycerol stock1 | LB(CP) |
INP-His-CBDcex glycerol stock2 | LB(CP) |
INP-His-CBDclos glycerol stock1 | LB(CP) |
INP-His-CBDclos glycerol stock2 | LB(CP) |
7/21
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/8 miniprep INP-His-scFv | 1 | DH5α /20 | LB(CP) |
2/8 miniprep BclA-His-scFv14 | 1 | DH5α /20 | LB(CP) |
2/24 miniprep BclA-His-CBDcex | 1 | DH5α /20 | LB(CP) |
2/24 miniprep BclA-His-CBDclos | 1 | DH5α /20 | LB(CP) |
2/25 miniprep INP-His-CBDcex | 1 | DH5α /20 | LB(CP) |
2/25 miniprep INP-His-CBDclos | 1 | DH5α /20 | LB(CP) |
7/22
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/4 parts awakening E0040 | 1 | DH5α /20 | LB(AMP) |
7/22
Liquid Culture
YamamotoName | medium |
---|---|
7/21 transformation BclA-His-scFv colony1 | LB(CP) |
7/21 transformation BclA-His-scFv colony2 | LB(CP) |
7/21 transformation INP-His-scFv colony1 | LB(CP) |
7/21 transformation INP-His-scFv colony2 | LB(CP) |
7/21 transformation BclA-His-CBDcex colony1 | LB(CP) |
7/21 transformation BclA-His-CBDcex colony2 | LB(CP) |
7/21 transformation BclA-His-CBDclos colony1 | LB(CP) |
7/21 transformation BclA-His-CBDclos colony2 | LB(CP) |
7/21 transformation INP-His-CBDcex colony1 | LB(CP) |
7/21 transformation INP-His-CBDcex colony2 | LB(CP) |
7/21 transformation INP-His-CBDclos colony1 | LB(CP) |
7/21 transformation INP-His-CBDclos colony 2 | LB(CP) |
7/25
Miniprep
YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
7/22 liquid culture BclA-His-scFv1 | 183.5 | 1.85 | 2.05 |
7/22 liquid culture BclA-His-scFv2 | 379.0 | 1.86 | 2.20 |
7/22 liquid culture INP-His-scFv1 | 189.6 | 1.85 | 2.21 |
7/22 liquid culture INP-His-scFv2 | 375.3 | 1.85 | 2.21 |
7/22 liquid culture BclA-His-CBDcex1 | 91.9 | 1.87 | 1.90 |
7/22 liquid culture BclA-His-CBDcex2 | 187.1 | 1.87 | 1.98 |
7/22 liquid culture BclA-His-CBDclos1 | 41.2 | 1.88 | 2.21 |
7/22 liquid culture BclA-His-CBDclos2 | 109.4 | 1.94 | 2.24 |
7/22 liquid culture INP-His-CBDcex1 | 71.0 | 1.73 | 1.19 |
7/22 liquid culture INP-His-CBDcex2 | 55.5 | 1.89 | 2.23 |
7/22 liquid culture INP-His-CBDclos1 | 88.6 | 1.82 | 1.61 |
7/22 liquid culture INP-His-CBDclos2 | 75.8 | 1.86 | 2.17 |
7/25
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
2/25 miniprep BclA-His-scFv1 | 10.9 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.1 | 30 |
2/25 miniprep BclA-His-scFv2 | 5.3 | 0 | 1 | 0 | 1 | 10×H /3 | 3.0 | 16.7 | 30 |
2/25 miniprep INP-His-scFv1 | 10.5 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.5 | 30 |
2/25 miniprep BclA-His-CBDcex2 | 10.7 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 14.3 | 30 |
2/25 miniprep BclA-His-CBDclos2 | 18.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 6.7 | 30 |
2/25 miniprep INP-His-CBDcex1 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
2/25 miniprep INP-His-CBDclos1 | 22.6 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 2.4 | 30 |
Samples of "2/25 miniprep BclA-His-scFv1" and "2/25 miniprep INP-His-scFv1" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡ is restriction enzyme).
7/26
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 7/25 restriction enzyme digestion BclA-His-scFv1 DraⅡcut | 30 |
3 | 7/25 restriction enzyme digestion BclA-His-scFv2 XPcut | 30 |
4 | 7/25 restriction enzyme digestion INP-His-scFv1 DraⅡcut | 30 |
5 | 7/25 restriction enzyme digestion BclA-His-CBDcex2 SPcut | 30 |
6 | 7/25 restriction enzyme digestion BclA-His-CBDclos2 SPcut | 30 |
7 | 7/25 restriction enzyme digestion INP-His-CBDcex1 SPcut | 30 |
8 | 7/25 restriction enzyme digestion INP-His-CBDclos1 SPcut | 30 |
![](https://static.igem.org/mediawiki/2016/a/af/T--Kyoto--7_26_No96_Gel_Extraction_Notsu_10_02_08_39_23.1.jpg
)
![](https://static.igem.org/mediawiki/2016/1/1a/T--Kyoto--
7_26_No96_Gel_Extraction_Notsu_10_02_08_39_23.2.jpg
)
7/26
Gel Extraction(Measurement)
YamamotoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
7/26 Gel Extraction BclA-His-scFv2 XPcut | 3.8 | 5.14 | 0.01 |
7/26 Gel Extraction BclA-His-CBDcex2 SPcut | 10.4 | 3.24 | 0.01 |
7/26 Gel Extraction BclA-His-CBDclos2 SPcut | 6.0 | 2.67 | 0.01 |
7/26 Gel Extraction INP-His-CBDcex1 SPcut | 8.1 | 2.03 | 0.02 |
7/26 Gel Extraction INP-His-CBDclos1 SPcut | 14.6 | 2.57 | 0.02 |
7/26
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
7/25 miniprep BclA-His-scFv1 | 10.9 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.1 | 30 |
7/25 miniprep BclA-His-scFv1 | 3.6 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 3.4 | 10 |
7/25 miniprep BclA-His-scFv1 | 3.6 | 0 | 0 | 0 | 0 | 0 | 0 | 6.4 | 10 |
7/25 miniprep BclA-His-scFv2 | 5.3 | 0 | 1 | 0 | 1 | 10×H /3 | 3.0 | 16.7 | 30 |
/25 miniprep INP-His-scFv1 | 10.5 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.5 | 30 |
/25 miniprep INP-His-scFv1 | 3.5 | 0 | 0 | 0 | 1 | 10×H /1 | 0 | 3.5 | 10 |
/25 miniprep INP-His-scFv1 | 3.5 | 0 | 0 | 0 | 0 | 0 | 0 | 6.5 | 10 |
7/25 miniprep BclA-His-CBDcex2 | 10.7 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 14.3 | 30 |
7/25 miniprep BclA-His-CBDclos2 | 18.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 6.7 | 30 |
7/25 miniprep INP-His-CBDcex2 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
7/25 miniprep INP-His-CBDclos2 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
Samples of "2/25 miniprep BclA-His-scFv(10.9µl)" and "2/25 miniprep INP-His-scFv1(10.5µl)" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡ is restriction enzyme).
7/26
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 DraⅡcut | 30 |
3 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 XPcut | 10 |
4 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 | 10 |
5 | 2/26Restriction Enzyme Digestion INP-His-scFv1 DraⅡcut | 30 |
6 | 2/26Restriction Enzyme Digestion INP-His-scFv1EPcut | 10 |
7 | 2/26Restriction Enzyme Digestion INP-His-scFv1 | 10 |
- | - | - |
1' | 1kb ladder | 5 |
2' | 2/26Restriction Enzyme Digestion BclA-His-scFv2 XPcut | 30 |
3' | 2/26Restriction Enzyme Digestion BclA-His-CBDcex2 SPcut | 30 |
4' | 2/26Restriction Enzyme Digestion BclA-His-CBDclos2 SPcut | 30 |
5' | 2/26Restriction Enzyme Digestion INP-His-CBDcex2 SPcut | 30 |
6' | 2/26Restriction Enzyme Digestion INP-His-CBDclos2 SPcut | 30 |
![](https://static.igem.org/mediawiki/2016/5/59/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-1.jpg
)
![](https://static.igem.org/mediawiki/2016/3/36/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-2.jpg)
![](https://static.igem.org/mediawiki/2016/a/a1/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.2-1.jpg
)
![](https://static.igem.org/mediawiki/2016/f/fe/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.2-2.jpg
)
7/27
Ligation
YamamotoInsert DNA(7/27purification) /µ? | Vector DNA(7/25purification) /µ? | Ligation High /µ? | Total |
---|---|---|---|
- | BclA-scFv1 DraⅡcut /7.5 | 7.5 | 15 |
- | INP-scFv1 DraⅡcut /7.5 | 7.5 | 15 |
BclAscFv2 XPcut /7.16 | BclA-CBDcex2 SPcut /0.34 | 7.5 | 15 |
BclAscFv2 XPcut /3.75 | BclA-CBDclos2 SPcut /3.75 | 7.5 | 15 |
BclAscFv2 XPcut /1.88 | INP-CBDcex2 SPcut /5.62 | 7.5 | 15 |
BclAscFv2 XPcut /5.0 | INP-CBDclos2 SPcut /2.5 | 7.5 | 15 |
7/27
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
BclAscFv1 DraⅡcut | 2 | DH5α /20 | LB(CP) |
INPscFv1 DraⅡcut | 2 | DH5α /20 | LB(CP) |
BclAscFv+BclACBDcex | 2 | DH5α /20 | LB(CP) |
BclAscFv+BclACBDclos | 2 | DH5α /20 | LB(CP) |
BclAscFv+INPCBDcex | 2 | DH5α /20 | LB(CP) |
BclAscfv+INPCBDclos | 2 | DH5α /20 | LB(CP) |
7/27
Gel Extraction(Measurement)
YamamotoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
7/27 Gel Extraction BclA-His-scFv1 DraⅡcut | 4.8 | -7.11 | 1.04 |
7/27 Gel Extraction INP-His-scFv1 DraⅡcut | 40.4 | 1.32 | 1.00 |
7/27 Gel Extraction BclA-His-scFv2 XPcut | 9.3 | -4.24 | 0.02 |
7/27 Gel Extraction BclA-His-CBDcex2 SPcut | 174.5 | 1.32 | 0.84 |
7/27 Gel Extraction BclA-His-CBDclos2 SPcut | 9.7 | 5.84 | 0.19 |
7/27 Gel Extraction INP-His-CBDcex2 SPcut | 3.7 | -2.82 | 0.14 |
7/27 Gel Extraction INP-His-CBDclos2 SPcut | 22.6 | 2.51 | 0.21 |
7/28
Restriction Enzyme Digestion
YamamotoDNA /µ? | EcoR1 /µ? | Xba1 /µ? | Spe1 /µ? | Pst1 /µ? | DraⅡ /µ? | Buffer /µ? | BSA /µ? | MilliQ /µ? | Total /µ? |
---|---|---|---|---|---|---|---|---|---|
BclAscFv2 /5.3 | 0 | 0 | 0 | 0 | 1 | 10×L /3 | 0 | 20.7 | 30 |
BclAscFv2 /5.3 | 0 | 1 | 0 | 1 | 0 | 10×H /3 | 3 | 16.7 | 30 |
INPscFv2 /5.3 | 0 | 0 | 0 | 0 | 1 | 10×L /3 | 0 | 20.7 | 30 |
BclAcex2 /10.7 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 14.3 | 30 |
BclAclos1 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
INPcex1,2 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
INPclos1,2 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
7/28
Gel Extraction
KanekoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
7/28 BclA-scFv2 XPcut |
0.5µg/µl
260/280 2.36
260/230 1.15
7/28
Transformation
KanekoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/22 scFv colony2 | 2 | DH5α /20 |
7/28
Ligation
YamamotoInsert DNA /(µl) | Vector DNA /(µl) | Ligation high /(µl) | Total /(µl) |
---|---|---|---|
- | 7/28 Restriction Enzyme BclA-scFv DraⅡcut /7.5 | 7.5 | 15 |
- | 7/28 Restriction Enzyme INP-scFv DraⅡcut /7.5 | 7.5 | 15 |
7/28
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
7/28 ligation BclA-scFv control | 2 | DH5α /20 | LB(CP) |
7/28 ligation INP-scFv control | 2 | DH5α /20 | LB(CP) |
2/22 miniprep BAN-scFv | - | DH5α /20 | LB(CP) |
7/28
Liquid Culture
YamamotoName | medium |
---|---|
BclA-scFv glycerol stock | LB(CP) |
7/21 transformation INP-scFv colony1 | LB(CP) |
7/21 transformation BclA-CBDcex colony1 | LB(CP) |
7/21 transformation BclA-CBDclos colony1 | LB(CP) |
7/21 transformation INP-CBDcex colony1 | LB(CP) |
7/21 transformation INP-CBDclos colony1 | LB(CP) |
7/21 transformation | LB(CP) |
8/1
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
7/28 transformaton BclA control colony4 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton BclA control colony5 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton BclA control colony6 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony4 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony5 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony6 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
8/1
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 1min | 1 |
Denature | 95℃ | 15sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Extension | 72℃ | 10sec | 30 |
Final Elogation | 72℃ | 5min | 1 |
8/1
Miniprep
YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
7/28 liquid culture BclA-scFv | 239.1 | 1.87 | 1.78 |
7/28 liquid culture INP-scFv colony1 | 263.4 | 1.86 | 1.99 |
7/28 liquid culture E0040 colony1 | 98.8 | 1.84 | 2.15 |
7/28 liquid culture BclA-CBDcex colony1 | 109.9 | 1.82 | 1.37 |
7/28 liquid culture BclA-CBDclos colony1 | 94.1 | 1.89 | 1.93 |
7/28 liquid culture INP-CBDcex colony1 | 111.4 | 1.82 | 1.39 |
7/28 liquid culture INP-CBDclos colony1 | 185.1 | 1.81 | 1.51 |
8/1
Electrophoresis
IguchiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 8/1 colonyPCR BclA control colony4 /10 | 672 |
2 | 8/1 colonyPCR BclA control colony5 /10 | 672 |
3 | 8/1 colonyPCR BclA control colony6 /10 | 672 |
4 | 8/1 colonyPCR INP control colony4 /10 | 1563 |
5 | 8/1 colonyPCR INP control colony5 /10 | 1563 |
6 | 8/1 colonyPCR INP control colony6 /10 | 1563 |
7 | 1kb ladder /5 |
![](https://static.igem.org/mediawiki/2016/8/83/T--Kyoto--8_1_No12_Electrophoresis_Notsu_09_30_08_29_26.1.jpg)
![](https://static.igem.org/mediawiki/2016/c/c9/T--Kyoto--8_1_No12_Electrophoresis_Notsu_09_30_08_29_26.2.jpg)
8/1
Restriction Enzyme Digestion
IguchiSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H 3 | 3 | 13.64 | 30 |
8/1 miniprep BclA-CBDcex colony1 | 18.2 | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
8/1 miniprep BclA-CBDclos colony1 | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
8/1 miniprep INP-CBDcex colony1 | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos colony1 | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | 30 |
8/2
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | BclA-scFv XPcut | 30 |
2 | BclA-CBDcex SPcut | 30 |
3 | BclA-CBDclos SPcut | 30 |
4 | INP-CBDcex SPcut | 30 |
5 | INP-CBDclos SPcut | 30 |
6 | 1kb ladder | 30 |
![](https://static.igem.org/mediawiki/2016/2/28/T--Kyoto--8_2_No14_Gel_Extraction_Notsu_09_30_08_40_44.1.jpg)
![](https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--8_2_No14_Gel_Extraction_Notsu_09_30_08_40_44.2.jpg)
8/2
Gel Extraction
YamamotoSample | Concentration ng/µl | 260/280 | 260/230 |
---|---|---|---|
8/2 Electrophoresis BclA-scFv XPcut | 1.0 | 23.20 | 0.12 |
8/2 Electrophoresis BclA-CBDcex SPcut | 10.8 | 1.89 | 1.35 |
8/2 Electrophoresis BclA-CBDclos SPcut | 18.0 | 1.92 | 1.88 |
8/2 Electrophoresis INP-CBDcex SPcut | 25.4 | 1.93 | 1.81 |
8/2 Electrophoresis INP-CBDclos SPcut | 60.9 | 1.23 | 1.05 |
8/2
Ligation
YamamotoVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
8/2 purification BclA-CBDcex SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification BclA-CBDclos SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification INP-CBDcex SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification INP-CBDclos SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2
Transformation
Yamamoto, YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/2 ligation BclA-scFv+BclA-CBDcex | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+BclA-CBDclos | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+INP-CBDcex | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+INP-CBDclos | 2 | DH5α /20 | LB(CP) |
8/2
Liquid Culture
YamadaName | medium |
---|---|
BclA-CBDcex glycerol stock | LB(CP) |
BclA-CBDclos glycerol stock | LB(CP) |
INP-CBDcex glycerol stock | LB(CP) |
INP-CBDclos glycerol stock | LB(CP) |
8/3
Liquid Culture
YamadaName | medium |
---|---|
7/28 transformation BclA control colony5 | LB(CP) |
7/28 transformation BclA control colony6 | LB(CP) |
7/28 transformation INP control colony4 | LB(CP) |
7/28 transformation INP control colony5 | LB(CP) |
7/28 transformation INP control colony6 | LB(CP) |
8/3
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H /3 | 3 | 13.64 | 30 |
8/1 miniprep BclA-CBDcex | 18.2 | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
8/1 miniprep BclA-CBDclos | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
8/1 miniprep INP-CBDcex | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | 30 |
8/3
Ligation
YamamotoVector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
8/1 Restriction Enzyme Digestion BclA-CBDcex /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion BclA-CBDclos /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion INP-CBDcex /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion INP-CBDclos /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/3
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/3 ligation BclA-scFv+BclA-CBDcex | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+BclA-CBDclos | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+INP-CBDcex | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+INP-CBDclos | 15 | DH5α /20 | LB(CP) |
8/4
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
8/3 transformation BclA-scFv+BclA-CBDcex colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/4
PCR (Steps)
YamamotoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 1min | 1 |
Denature | 95℃ | 15sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Extension | 72℃ | 10sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
8/4
Miniprep
YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/3 liquid culture BclA control colony5 | 137.2 | 1.77 | 1.03 |
8/3 liquid culture BclA control colony6 | 117.6 | 1.87 | 1.54 |
8/3 liquid culture INP control4 | 165.2 | 1.87 | 1.68 |
8/3 liquid culture INP control5 | 150.6 | 1.90 | 1.81 |
8/3 liquid culture INP control6 | 147.4 | 1.79 | 1.63 |
8/4
Sequence
Yamamoto, YamadaNo | primer(10µM) | MilliQ | Total |
---|---|---|---|
1 / 2 | BclA_5_VF(2.18µl) / BclA_5_VR(2.18µl) | 5.82µl / 5.82µl | 10µl |
3 / 4 | BclA_6_VF(2.55µl) / BclA_6_VR(2.55µl) | 5.45µl / 5.45µl | 10µl |
5 / 6 | INP_4_VF(1.81µl) / INP_4_VR(1.81µl) | 6.19µl / 6.19µl | 10µl |
7 / 8 | INP_5_VF(1.99µl) / INP_5_VR(1.99µl) | 6.01µl / 6.01µl | 10µl |
8/4
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony1 /10 | 1716 |
2 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony2 /10 | 1716 |
3 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony3 /10 | 1716 |
4 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony4 /10 | 1716 |
5 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony1 /10 | 1716 |
6 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony2 /10 | 1716 |
7 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony3 /10 | 1716 |
8 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony4 /10 | 1716 |
9 | 100bp ladder /5 | - |
10 | 1kb ladder /5 | - |
11 | - | - |
12 | - | - |
![](https://static.igem.org/mediawiki/2016/7/7f/T--Kyoto--8_4_No26_Electrophoresis_Notsu_10_01_11_15_31.jpg)
8/4
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony1 /10 | 2606 |
2 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony2 /10 | 2606 |
3 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony3 /10 | 2606 |
4 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony4 /10 | 2606 |
5 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony1 /10 | 2606 |
6 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony2 /10 | 2606 |
7 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony3 /10 | 2606 |
8 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony4 /10 | 2606 |
9 | 100bp ladder /5 | - |
10 | 1kb ladder /5 | - |
11 | - | - |
12 | - | - |
![](https://static.igem.org/mediawiki/2016/c/cc/T--Kyoto--8_4_No27_Electrophoresis_Notsu_10_01_11_18_45.jpg)
8/5
PartsAwaking
YamamotoDescription | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
---|---|---|---|---|---|---|
RFP | J23105 | J61002 | 17N | 2016 | 4 | 10 |
8/5
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/5 Parts awakening J23105 | 1µl | DH5α /20 | LB(AMP) |
8/9
Restriction Enzyme Digestion
Iguchi, YamadaSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H /3 | 3 | 13.69 | 30 |
8/1 and 7/25 miniprep BclA-CBDcex | 18. | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
2/25 miniprep BclA-CBDclos | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
2/25 and 7/25 minniprep INP-CBDcex | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | 30 |
8/9
Miniprep
YamadaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/3 liquid culture BclA-CBDccex | 225.8 | 1.74 | 1.63 |
8/3 liquid culture BclA-CBDclos | 102.3 | 1.80 | 1.66 |
8/3 liquid culture INP-CBDcex | 112.4 | 1.86 | 1.77 |
8/3 liquid culture INP-CBDclos | 42.8 | 1.76 | 0.78 |
8/10
Gel Extraction
YamadaLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
A1 | 1kb ladder | 10 |
A2 | BclA-scFv 8/9XPcut | 30 |
A3 | BclA-CBDcex 8/9 SPcut | 30 |
A4 | BclA-CBDclos 8/9 SPcu | 30 |
- | - | - |
B1 | 1kb ladder | 10 |
B2 | INP-CBDcex 8/9 SPcut | 30 |
B3 | INP-CBDclos 8/9 SPcut | 30 |
![](https://static.igem.org/mediawiki/2016/b/be/T--Kyoto--8_10_No31_Gel_Extraction_Notsu_10_01_11_59_14.1.jpg)
![](8_10_No31_Gel_Extraction_Notsu_10_01_11_59_14.2.jpg
https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--)
8/10
Gel Extraction(Measurement)
YamadaSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
BclA-scFv 8/9XPcut | 2.6 | 1.13 | 0.58 |
BclA-CBDcex 8/9 SPcut | 6.3 | 1.54 | 0.97 |
BclA-CBDclos 8/9 SPcut | 4.7 | 1.49 | 0.48 |
INP-CBDcex 8/9 SPcut | 4.4 | 1.39 | 0.67 |
INP-CBDclos 8/9 SPcut | 3.5 | 1.29 | 0.77 |
8/10
Miniprep
NotsuDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/9 liquid culture J23105 (AMP) RFP colony3 | 178.8 | 1.98 | 1.92 |
8/12
Liquid Culture
IguchiName | medium |
---|---|
8/2 liquid culture INP-CBDclos | LB(CP) |
BclA-scFv glycerol stock 1 | LB(CP) |
BclA-scFv glycerol stock 2 | LB(CP) |
BclA-scFv glycerol stock 3 | LB(CP) |
BclA-scFv glycerol stock 4 | LB(CP) |
8/18
Miniprep
NotsuDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/12 liquid culture INP-CBDclos | 45.3 | 1.82 | 1.31 |
8/12 liquid culture BclA-scFv glycerol stock 1 | 101.6 | 1.59 | 0.71 |
8/12 liquid culture BclA-scFv glycerol stock 2 | 100.7 | 1.67 | 0.78 |
8/12 liquid culture BclA-scFv glycerol stock 3 | 77.8 | 1.60 | 0.70 |
8/12 liquid culture BclA-scFv glycerol stock 4 | 41.0 | 1.87 | 1.27 |
8/20
Liquid Culture
Ohara, IsodaName | medium |
---|---|
BclA-CBDcex | LB(CP) |
BclA-CBDclos | LB(CP) |
INP-CBDcex | LB(CP) |
INP-CBDclos | LB(CP) |
8/21
Liquid Culture
OharaName | medium |
---|---|
BclA-scFv [1](glisto) | LB(CP) |
BclA-scFv [2] | LB(CP) |
BclA-scFv [3] | LB(CP) |
BclA-scFv [4] | LB(CP) |
8/22
Electrophoresis
SassaLane | sample/(µl) | Length(bp) |
---|---|---|
8/22 Restraction Ensyme Digestion 1.1kb ladder | 5 | |
8/22 Restraction Ensyme Digestion Bcla-cex | 25 | 2520 |
8/22 Restraction Ensyme Digestion Bcla-clos | 25 | 2520 |
8/22 Restraction Ensyme Digestion INP-cex(s,pcut) | 25 | 3500 |
8/22 Restraction Ensyme Digestion INP-clos | 25 | 3500 |
8/22 Restraction Ensyme Digestion INP-cex | 25 | 2541 |
8/22 Restraction Ensyme Digestion J23105 (AMP) RFP 8/21 old | 25 | 950 |
8/22 Restraction Ensyme Digestion Liquid culture Bcla-scfv glisto 4 | 25 | 932 |
8/22 Restraction Ensyme Digestion Bcla-scfv glisto 2 | 25 | 932 |
100ladder | 5 |
![](https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.1.jpg)
![](https://static.igem.org/mediawiki/2016/a/a4/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.2.jpg)
![](https://static.igem.org/mediawiki/2016/f/fa/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.3.jpg)
8/22
Restriction Enzyme Digestion
OharaSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/12 Liquid cultureBclA-scFv glisto [4] | 38 | 0 | 1 | 0 | 1 | 5 | 5 | 0 | 50 |
8/12 Liquid cultureBclA-scFv glisto [2] | 38 | 0 | 1 | 0 | 1 | 5 | 5 | 0 | 50 |
8/22
Transformation
OharaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/9 J23105 RFP | DH5α /20 | LB(AMP) |
8/22
CBD 1st
Yoshimotosample | before | after | wash |
---|---|---|---|
BclAclos+ | 0.208/0.206/0.206 | 0.131/0.135/0.14 | 0.111/0.112/0.109 |
INPclos+ | 0.183/0.186/0.184 | 0.239/0.23/0.22 | 0.158/0.155/0.169 |
8/22
CBD 2nd
Yoshimotosample | before | after | wash |
---|---|---|---|
BclAclos+ | 0.208/0.205/0.204 | 0.078/0.074/0.075 | 0.056/0.056/0.039 |
INPclos+ | 0.183/0.181/0.187 | 0.093/0.097/0.099 | 0.067/0.073/0.069 |
8/23
CBD 3rd
Yamadasample | before | after | wash |
---|---|---|---|
BclAcex+ (15min) | 0.247/0.247/0.248 | 0.221/0.224/0.218 | 0.085/0.075/0.073 |
BclAcex+ (30min) | 0.247/0.247/0.248 | 0.243/0.229/0.228 | 0.034/0.048/0.038 |
BclAcex+ (60min) | 0.247/0.247/0.248 | 0.224/0.221/0.217 | 0.065/0.064/0.065 |
BclAcex- (15min) | 0.247/0.247/0.248 | 0.257/0.253/0.258 | |
BclAcex- (30min) | 0.247/0.247/0.248 | 0.236/0.230/0.235 | |
BclAcex- (60min) | 0.247/0.247/0.248 | 0.249/0.248/0.247 | |
cellulose only (60min) | 0.021/0.021/0.023 |
8/23
CBD 4th
Yamadasample | before | after | wash |
---|---|---|---|
BclAcex+ (2ml tube) | 0.227/0.226/0.225 | 0.108/0.108/0.108 | 0.076/0.074/0.081 |
BclActl+ (2ml tube) | 0.186/0.203/0.189 | 0.082/0.081/0.082 | 0.055/0.046/0.044 |
BclAcex- (2ml tube) | 0.235/0.237/0.233 | 0.261/0.262/0.254 | |
BclAcex+ (1.5ml tube) | 0.237/0.226/0.236 | 0.159/0.157/0.158 | 0.066/0.063/0.065 |
BclActl+ (1.5ml tube) | 0.177/0.173/0.179 | 0.112/0.109/0.113 | 0.080/0.076/0.095 |
BclAcex- (1.5ml tube) | 0.240/0.235/0.238 | 0.255/0.254/0.253 |
8/24
Liquid Culture
OharaName | medium |
---|---|
8/24 BclA-scFv 1 | LB(CP) |
8/24 BclA-scFv 2 | LB(CP) |
8/24 BclA-scFv 3 | LB(CP) |
8/24 BclA-scFv 4 | LB(CP) |
8/29
Miniprep
SassaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
1. 8/23Liquidculture Bcla scFv | 111.6 | 1.98 | 1.82 |
2. 8/23Liquidculture Bcla scFv | 143.1 | 2.00 | 2.03 |
2'. 8/23Liquidculture Bcla scFv | 141.6 | 2.04 | 2.10 |
3. 8/23Liquidculture Bcla scFv | 145.2 | 2.07 | 2.01 |
8/23Liquidculture J23105 | 63.1 | 2.01 | 1.69 |
8/23Liquidculture J23105 | 68.1 | 2.00 | 1.75 |
8/24
CBD 5th
Isodasample | before | after | wash |
---|---|---|---|
INPcex+ | 0.195/0.191/0.192 | 0.103/0.102/0.109 | 0.059/0.051/0.055 |
INPcex- | 0.190/0.191/0.205 | 0.186/0.187/0.186 | |
INPclos+ | 0.187/0.187/0.188 | 0.121/0.122/0.126 | 0.051/0.058/0.059 |
INPclos- | 0.196/0.195/0.191 | 0.206/0.204/0.202 | |
INPctl+ | 0.172/0.163/0.162 | 0.116/0.177/0.124 | 0.066/0.063/0.062 |
INPctl- | 0.171/0.175/0.165 | 0.167/0.182/0.171 |
8/24
CBD 6th
Isodasample | before | after | wash |
---|---|---|---|
INPcex+ | 0.184/0.184/0.189 | 0.104/0.106/0.106 | 0.044/0.044/0.056 |
INPcex- | 0.186/0.190/0.184 | 0.182/0.181/0.184 | |
INPclos+ | 0.184/0.189/0.185 | 0.097/0.098/0.099 | 0.037/0.037/0.038 |
INPclos- | 0.187/0.192/0.190 | 0.185/0.184/0.189 | |
INPctl+ | 0.173/0.184/0.180 | 0.095/0.099/0.095 | 0.045/0.046/0.046 |
INPctl- | 0.185/0.184/0.183 | 0.175/0.175/0.175 | |
BclAcex+ | 0.170/0.168/0.174 | 0.099/0.094/0.092 | 0.037/0.032/0.036 |
BclAcex- | 0.186/0.189/0.171 | 0.164/0.167/0.164 | |
cellulose only | 0.006/0.005/0.006 | 0.013/0.010/0.006 |
8/25
CBD 7th
Yamadasample | before | after | wash |
---|---|---|---|
BclAcex+ | 0.181/0.181/0.181 | 0.192/0.196/0.195 | 0/0/0 |
BclAcex- | 0.149/0.150/0.151 | 0.221/0.213/0.211 | |
BclActl+ | 0.169/0.165/0.167 | 0.154/0.156/0.157 | 0.012/0.013/0.011 |
BclActl- | 0.151/0.153/0.154 | 0.187/0.178/0.173 |
8/26
PCR (Target)
SassaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
8/10 miniprep J23105 /3 | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/24 OD0.206 INP-CBDclos /3 | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/24 OD0.202 INP-CBDcex | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/26
Electrophoresis
Yamamoto, SassaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder /5 | |
2 | 8/26 PCR INP-CBDcex VFVR /15 | |
3 | 8/26 PCR INP-CBDclos VFVR /15 |
![](https://static.igem.org/mediawiki/2016/4/4b/T--Kyoto--8_26_No62_Electrophoresis_Notsu_10_01_02_14_38.jpg)
8/26
Miniprep
SassaDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/26 Gel Extraction Bcla cex | 8.3 | 1.52 | 0.25 |
8/26 Gel Extraction Bcla clos | 10.2 | 1.70 | 0.26 |
8/26 Gel Extraction INP cex | 11.4 | 1.76 | 0.15 |
8/26 Gel Extraction INP clos | 7.7 | 1.62 | 0.09 |
8/26 Gel Extraction Bcla scFv glisto | 7.0 | 1.59 | 0.20 |
8/26 Gel Extraction Bcla scFv glisto | 14.1 | 6.33 | 0.04 |
8/26
CBD 8th
Yoshimotosample | before | after | wash |
---|---|---|---|
BclAcex+ | 0.183/0.185/0.188 | 0.116/0.115/0.114 | 0.086/0.086/0.089 |
BclActl+ | 0.116/0.115/0.114 | 0.096/0.092/0.087 | 0.09/0.079/0.084 |
cellulose only | 0.037/0.035/0.037 |
8/26
CBD 9th
Oharasample | before | after | wash |
---|---|---|---|
BclAcex+ | 0.186/0.186/0.184 | 0.170/0.169/0.170 | 0.021/0.024/0.020 |
BclAcex- | 0.154/0.149/0.149 | 0.142/0.143/0.141 | |
BclActl+ | 0.194/0.189/0.189 | 0.167/0.164/0.159 | 0.010/0.009/0.008 |
BclActl- | 0.162/0.168/0.169 | 0.159/0.162/0.165 |
8/29
Ligation
SassaInsert DNA | vector DNA | Ligation High | Total |
---|---|---|---|
8/26 Gel Extraction BclAscFv /3.4 | 8/26 Gel Extraction Bcla CBDcex /4.1 | 7.5 | 15 |
8/26 Gel Extraction BclAscFv /3.1 | 8/26 Gel Extraction Bcla CBD clos /4.4 | 7.5 | 15 |
8/26 Gel Extraction BclAscFvBclAscFv /3.8 | 8/26 Gel Extraction Bcla CBD cex /4.7 | 7.5 | 15 |
8/26 Gel Extraction BclAscFv /2.6 | 8/26 Gel Extraction Bcla CBD clos /4.9 | 7.5 | 15 |
8/29
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
![](https://static.igem.org/mediawiki/2016/0/0e/T--Kyoto--8_29_No68_General_Miyazaki_10_17_05_42_18.jpg)
blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
8/29
PCR (Target)
MiyazakiTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
BclA-CBDcex | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
BclA-CBDclos | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
INP-CBDcex | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
INP-CBDclos | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
![](https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--8_29_No69_PCR_%28Target%29_Notsu_10_01_02_11_53.jpg)
8/29
ligation
MiyazakiInsert DNA | Vector DNA | Ligation High | Total |
---|---|---|---|
BclAscFv XPcut 6.3 | INPCBDcex SPcut 1.2 | 7.5 | 15 |
BclAscFv XPcut 6.4 | INPCBDclos SPcut 1.1 | 7.5 | 15 |
8/29
Miniprep
Sassa, MiyazakiDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
BclA scFv 1 | 8.4 | 1.63 | 0.11 |
BclA scFv 2 | 5.6 | 1.91 | 0.04 |
INP CBDcex | 44.8 | 1.90 | 0.70 |
INP CBDclos | 33.1 | 1.82 | 0.68 |
BclA cex | 26.3 | 1.93 | 0.62 |
BclA clos | 24.4 | 1.93 | 0.66 |
J23105 VF VR | 33.2 | 1.88 | 0.24 |
9/5
Restriction Enzyme Digestion
MiyazakiSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/10 J23105 RFP miniprep | 20 | 0 | 1 | 0 | 1 | 10×H 3 | 3 | 2 | 30 |
9/5
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/29 Ligation Bclascfv-BclaCBDcex | 2 | CH5a/20 | LB(CP) |
〃Bclascfv-BclaCBDclos | 2 | 〃 | 〃 |
〃Bclascfv-INPCBDcex | 2 | 〃 | 〃 |
〃Bclascfv-INPCBDclos | 2 | 〃 |
9/5
Electrophoresis
YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 8/20 SPcut Bcla-CBDcex/25 | 2520 |
2 | 〃Bcla-CBDclos/25 | 〃 |
3 | 〃INP-CBDcex/25 | 〃 |
4 | 〃INP-CBDclos/25 |
![](https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--9_5_No74_Electrophoresis_Yamamoto_09_30_10_51_18.jpg)
9/5
Electrophoresis
MiyazakiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 100bp ladder/5 | 6×loading Dye - |
2 | 8/10 miniprep 9/5 xpcut J23105RFP/30 |
9/5
Gel Extraction
Sassa, MiyazakiLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
9/5 electrophoresis 2 | J23105 RFP XPcut backbone | |
9/5 electrophoresis 2 | J23105 RFP XPcut insert | |
9/5 electrophoresis 1 | BclAcex SPcut | |
9/5 electrophoresis 2 | BclAclos SPcut | |
9/5 electrophoresis 3 | INPcex SPcut | |
9/5 electrophoresis 4 | INPclos SPcut |
9/6
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
Bclascfv+BclaCBDcex colony1 | vf1.25 vr1.25 | 10 | KAPA2GFAST 12.5 | 25 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+BclaCBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+INPCBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+INPCBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
9/6
Ligation
YamamotoInsert DNA(µl) | Vector DNA/(µl) | Ligation High/(µl) | Total/(µl) |
---|---|---|---|
9/5 Gel Extraction BclA cex SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction BclA clos SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction INP cex SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction INP clos SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/6
Electrophoresis
YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/5 | 0 |
2 | 9/6 PCR scfv+Bclacex colony1/15 | 0 |
3 | 〃2/15 | 0 |
4 | 〃3/15 | 0 |
5 | 〃4/15 | 0 |
6 | 9/6 PCR scfv+Bclaclos colony1 | 0 |
7 | 〃2 | 0 |
8 | 〃3 | 0 |
9 | 〃4 | 0 |
10 | 9/6 PCR scfv+INPcex colony1 | 0 |
11 | 〃2 | 0 |
12 | 〃3 | 0 |
13 | 〃4 | 0 |
14 | 9/6 PCR scfv+INPclos colony1 | 0 |
15 | 〃2 | 0 |
16 | 〃3 | 0 |
17 | 〃4 | 0 |
![](https://static.igem.org/mediawiki/2016/a/a4/T--Kyoto--9_6_No81_Electrophoresis_Kosaka_09_30_11_22_50.jpg)
9/6
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
![](https://static.igem.org/mediawiki/2016/3/33/T--Kyoto--9_6_No82_General_Miyazaki_10_17_05_43_53.jpg)
blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
9/6
CBD 10th
Isodasample | before | after | wash |
---|---|---|---|
BclAcex+ | 0.239 | 0.222 | |
BclActl+ | 0.212 | 0.190 | |
BclAcex- | 0.239 | 0.233 | |
BclActl- | 0.221 | 0.210 |
9/6
CBD 11th
Isodasample | before | after | wash |
---|---|---|---|
BclAcex+ (NaCl) | 0.202 | 0.176 | |
BclAcex- (NaCl) | 0.202 | 0.206 | |
BclAcex- (NaCl) | 0.202 | 0.264 | |
BclAcex+ (PBS buffer) | 0.212 | 0.179 | |
BclAcex- (PBS buffer) | 0.212 | 0.209 | |
BclActl+ (PBS buffer) | 0.211 | 0.172 |
9/7
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
Bclascfv-BclaCBDcex colony5 | vf12.5 vr12.5 | 10 | KAPA2GFASTHSRM 12.5 | 25 |
〃6 | = | = | = | = |
〃7 | = | = | = | = |
〃8 | = | = | = | = |
〃9 | = | = | = | = |
〃10 | = | = | = | = |
9/5 Bclascfv-BclaCBDclos 5 | = | = | = | = |
〃6 | = | = | = | = |
9/5 Bclascfv-INPCBDcex1 | = | = | = | = |
〃2 | = | = | = | = |
〃3 | = | = | = | = |
〃4 | = | = | = | = |
Bclascfv-INPCBDclos1 | = | = | = | = |
〃2 | = | = | = | = |
〃3 | = | = | = | = |
〃4 | = | = | = | = |
![](https://static.igem.org/mediawiki/2016/c/c6/T--Kyoto--9_7_No83_Colony_PCR_%28typeA%29_Kosaka_09_30_11_33_11.jpg)
9/7
Miniprep
YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
Bcla-CBDcex | 99.7 | 1.94 | 2.08 |
Bcla-CBDclos | 73.5 | 1.96 | 2.15 |
INP-CBDcex | 45.3 | 1.99 | 2.14 |
INP-CBDclos | 49.6 | 1.89 | 1.89 |
9/7
Restriction Enzyme Digestion
YamamotoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
8/5 miniprep INP-CBDcex | 25 | 0 | 0 | 1 | 1 | 10×M/3 | 0 | 0 | 30 |
8/5 miniprep INP-CBDclos | 25 | 0 | 0 | 1 | 1 | 10×M/3 | 0 | 0 | 30 |
9/13 miniprep BclA-scFv1 | 22 | 0 | 1 | 0 | 1 | 10×H/3 | 3 | 0 | 30 |
9/7
Liquid Culture
YamamotoName | medium |
---|---|
9/5 Transformation BclascFv-BclaCBDcex colony5 | LB(CP)/5ml |
〃BclascFv-BclaCBDclos colony4 | LB(CP)/5ml |
9/7
SEM
Oharanumber | sample | OD600 | VLP(µl) |
---|---|---|---|
1 | BclAscFv 3c5 | 0.990 | 1 |
2 | BclAscFv 1c5 | 1.001 | 1 |
3 | INPscFv 3c5 | 1.062 | 1 |
4 | BclA CTL | 1.053 | 1 |
5 | INP CTL | 0.952 | 1 |
9/7
CBD 12th
Isodasample | before | after | wash |
---|---|---|---|
BclAcex+ (PBS buffer) | 0.195/0.192 | 0.188/0.189 | |
BclActl+ (PBS buffer) | 0.198/0.195 | 0.192/0.189 | |
BclAcex+ (CB buffer) | 0.186/0.184 | 0.176/0.173 | |
BclActl+ (CB buffer) | 0.172/0.169 | 0.151/0.155 |
9/7
CBD 13th
Isodasample | before | after | wash |
---|---|---|---|
BclAcex+ (PBS buffer) (Bemliese) | 0.201/0.201 | 0.201/0.201 | |
BclActl+ (PBS buffer) (Bemliese) | 0.224/0.223 | 0.218/0.215 | |
BclAcex+ (CB buffer) (Bemliese) | 0.266/0.257 | 0.243/0.242 | |
BclActl+ (CB buffer) (Bemliese) | 0.178/0.177 | 0.157/0.149 | |
BclAcex+ (PBS buffer) (Kimwipe) | 0.203/0.201 | 0.200/0.196 | |
BclActl+ (PBS buffer) (Kimwipe) | 0.221/0.220 | 0.224/0.229 | |
BclAcex+ (CB buffer) (Kimwipe) | 0.273/0.266 | 0.244/0.242 | |
BclActl+ (CB buffer) (Kimwipe) | 0.172/0.172 | 0.154/0.154 |
9/8
Gel Extraction
YamamotoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 9/7 restriction enzyme design BclA scFv X/Pcut | 30 |
3 | 9/7 restriction enzyme design INP CBDcex S/Pcut | 30 |
4 | 9/7 restriction enzyme design INP CBDclos S/Pcut | 30 |
5 | 100bp ladder | 5 |
![](https://static.igem.org/mediawiki/2016/a/a8/T--Kyoto--9_8_No88_Gel_Extraction_Miyazaki_10_15_12_18_19.1.jpg)
![](https://static.igem.org/mediawiki/2016/2/2a/T--Kyoto--9_8_No88_Gel_Extraction_Miyazaki_10_15_12_18_19.2.jpg)
9/8
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
9/5 TransformationINPcex-scFv colony5 | vf 1.25 vr1.25 | 10 | KAPA2GFAST/12.5 | 25 |
〃colony6 | = | = | = | = |
〃colony7 | = | = | = | = |
〃colony8 | = | = | = | = |
9/5 Transformation INPclos-BclascFv colony1 | = | = | = | = |
〃colony2 | = | = | = | = |
〃colony5 | = | = | = | = |
〃colony6 | = | = | = | = |
![](https://static.igem.org/mediawiki/2016/2/26/T--Kyoto--9_8_No89_Colony_PCR_%28typeA%29_Kosaka_10_02_01_52_26.jpg)
9/8
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
![](https://static.igem.org/mediawiki/2016/6/64/T--Kyoto--9_8_No90_General_Miyazaki_10_17_05_45_26.jpg)
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
9/8
Ligation
YamamotoInsert/µl | Vector/µl | Ligation High/µl | Total |
---|---|---|---|
9/8 Gel Extraction BclA scFv X/Pcut /6 | 9/8 Gel Extraction INP CBDcex S/Pcut /1.5 | 7.5 | 15 |
9/8 Gel Extraction BclA scFv X/Pcut /6 | 9/8 Gel Extraction INP CBDcex S/Pcut /1.5 | 7.5 | 15 |
9/8
Transformation
Yamamoto, SassaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
9/8 Ligation Bclascfv-INPCBDcex | 2 | DH5a/20 | LB(CP) |
〃BclascFv-INPCBDclos | 2 | 〃 | 〃 |
9/7Miniprep BclaCBDcex+8/24 Miniprep J23105 | 1 | 〃 | LB(CP+AMP) |
〃Bcla-CBDclos+8/24 J23105 | 1 | 〃 | 〃 |
9/7 Miniprep INP-CBDcex+8/24 Miniprep J23105 | 1 | 〃 | 〃 |
9/7 Miniprep INP-CBDclos+8/24 Miniprep J23105 | 1 | 〃 | 〃 |
9/8
CBD 14th
Isodasample | before | after | wash |
---|---|---|---|
CBDcex+ (CB buffer) | 0.241/0.236 | 0.215/0.212 | |
CBDctl+ (CB buffer) | 0.182/0.180 | 0.159/0.159 | |
BclAcex+ (CB buffer + Na2CO3) | 0.235/0.235 | 0.177/0.177 | |
BclActl+ (CB buffer+Na2CO3) | 0.227/0.218 | 0.155/0.156 |
9/9
Colony PCR (typeA)
IguchiTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INPcex-scFv colony9 | vf1.25 vr1.25 | 8.5 | KAPA2GFAST 12.5 | 25 |
〃colony10 | 〃 | 〃 | 〃 | 〃 |
〃colony11 | 〃 | 〃 | 〃 | 〃 |
9/5 transformation INPclos-scFv colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony5 | 〃 | 〃 | 〃 | 〃 |
![](https://static.igem.org/mediawiki/2016/0/02/T--Kyoto--9_9_No96_Colony_PCR_%28typeA%29_Kosaka_10_02_02_29_44.jpg)
9/9
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 1µl |
4 | Sano VLP 0.2µl |
5 | Sano VLP 0.1µl |
6 | Sano VLP 0.05µl |
7 | Sano VLP 0.02µl |
8 | Sano VLP 0.5µl 95℃ without boiling |
9 | only SDS lysis buffer |
![](https://static.igem.org/mediawiki/2016/7/7a/T--Kyoto--9_9_No98_General_Miyazaki_10_17_05_52_42.jpg)
1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h
9/9
Miniprep
MiyazakiDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
scFv Bcla/1ul | 271.8 | 2.09 | 2.15 |
VLP/1ul | 1.67 | 1.98 | - (A-280 10mmPath 1.670) |
9/9
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
BclaCBDcexJ23105 | vf1.25 vr1.25 | 10 | KAPA2GFAST HSRM withDye/12.5 | 25 |
9/6 Transformation BclaCBDclos J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcex J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcexJ23105 | 〃 | 〃 | 〃 | 〃 |
![](https://static.igem.org/mediawiki/2016/1/1b/T--Kyoto--9_9_No100_Colony_PCR_%28typeA%29_Kosaka_10_02_02_51_54.jpg)
9/9
Transformation
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/24 miniprep Bcla-his-cex colony2 | 0.1 | DH5a/20ul | LB(CP) |
9/9
Liquid Culture
IguchiName | medium |
---|---|
9/5 transformation INPcex-BclascFv colony9 | LB(CP)/5ml |
9/5 transformation INPclos-BclascFv colony1 | LB(CP)/5ml |
9/9
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
9/5 transformation Bcla CBDcex J23105 | vf1.0 vr1.0 | 23 | Quick Taq/25 | 50 |
〃BclaCBDclos J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcex J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDclos J23105 | 〃 | 〃 | 〃 | 〃 |
9/12
Restriction Enzyme Digestion
MiyazakiSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
9/9miniprep scFv Bcla lc3 | 29ul | 1 | 0 | 1 | 10×H/5 | 5 | 9 | 50 |
9/12
Electrophoresis
MiyazakiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/5 | - |
2 | Bcla CBDcex j23105/15[1] | 789 1140 |
3 | 〃[2] | 〃 |
4 | Bcla CBDclos J23105[1] | 757 1140 |
5 | 〃[2] | 〃 |
6 | INP CBDcex J23105[1] | 1664 1140 |
7 | 〃[2] | 〃 |
8 | INP CBDclos J23105[1] | 1796 1140 |
9 | 〃[2] | 〃 |
10 | 100bp ladder | 〃 |
![](https://static.igem.org/mediawiki/2016/4/4f/T--Kyoto--9_12_No105_Electrophoresis_Kosaka_10_02_03_10_40.jpg)
9/12
Colony PCR (typeA)
MiyazakiTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
Bcla CBDcex J23105[1] | vf0.5 vr0.5 | 11.5 | Quick Taq/12.5 | 25 |
〃[2] | 〃 | 〃 | 〃 | 〃 |
〃[3] | 〃 | 〃 | 〃 | 〃 |
〃[4] | 〃 | 〃 | 〃 | 〃 |
Bcla CBDclos J23105[1] | 〃 | 〃 | 〃 | 〃 |
〃[2] | 〃 | 〃 | 〃 | 〃 |
〃[3] | 〃 | 〃 | 〃 | 〃 |
〃[4] | 〃 | 〃 | 〃 | 〃 |
9/12
Colony PCR (typeA)
MiyazakiTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INP CBDcex J23105 | vf0.5 vr0.5 | 11.5 | Quick Taq12.5 | 25 |
INP〃[2] | = | = | = | = |
〃[3] | = | = | = | = |
〃[4] | = | = | = | = |
〃clos[1] | = | = | = | = |
〃[2] | = | = | = | = |
〃[3] | = | = | = | = |
〃[4] | = | = | = | = |
9/13
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 1µl |
3 | Sano VLP 1µl |
4 | Sano VLP 0.2µl |
5 | Sano VLP 0.1µl |
6 | Sano VLP 0.05µl |
7 | Sano VLP 0.02µl |
8 | Sano VLP 0.5µl 95℃ without boiling |
9 | only SDS lysis buffer |
![](https://static.igem.org/mediawiki/2016/c/c8/T--Kyoto--9_13_No112_General_Miyazaki_10_17_05_58_13.1.jpg)
![](https://static.igem.org/mediawiki/2016/3/3c/T--Kyoto--9_13_No112_General_Miyazaki_10_17_05_58_13.2.jpg)
1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h
30ml TBS-T/blocking reagent 1.5g
9/14
Liquid Culture
IsodaName | medium |
---|---|
9/9 transformation BclA-CBDcex | LB(CP) |
8/24 transformation BclA-CBDclos | LB(CP) |
8/24 transformation INP-CBDcex | LB(CP) |
8/20 transformation | LB(CP) |
9/14
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl+INP-scFv 100µl |
3 | Sano VLP 0.5µl+BAN-scFv 100µl |
4 | Sano VLP 0.2µl +BAN-scFv 100µl |
5 | Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl |
6 | Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl |
7 | Sano VLP 0.2µl +BANcontrol100µl |
8 | Sano VLP 0.2µl 95℃ without boiling |
9 | Sano VLP 0.5µl 95℃ without boiling |
10 | Marker |
![](https://static.igem.org/mediawiki/2016/6/6a/T--Kyoto--9_14_No114_General_Miyazaki_10_17_06_06_07.jpg)
1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g
9/15
Western Blotting
Ohara, Sassalane | sample |
---|---|
1 | Marker |
2 | Sano VLP 0.5µl+INP-scFv 100µl |
3 | Sano VLP 0.5µl+BAN-scFv 100µl |
4 | Sano VLP 0.2µl +BAN-scFv 100µl |
5 | Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl |
6 | Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl |
7 | Sano VLP 0.2µl +BANcontrol100µl |
8 | Sano VLP 0.2µl 95℃ without boiling |
9 | Sano VLP 0.5µl 95℃ without boiling |
10 | Marker |
![](https://static.igem.org/mediawiki/2016/8/8f/T--Kyoto--9_15_No116_General_Miyazaki_10_17_06_11_34.jpg)
1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g
9/15
Liquid Culture
SassaName | medium |
---|---|
9/7 BclA scFv 3C5 9/7 2 | LB(CP) /20mL |
9/14 BclA cex (9/9 transformation) | LB(CP) /20mL |
9/7 INP scFv 3C5 | LB(CP) /20mL |
9/9 BclA scFv 1c3 | LB(CP) /20mL |
9/7 INP CTL | LB(CP) /20mL |
9/7 BclA CTL 2 | LB(CP) /20mL |
9/15
Liquid Culture
Ohara, IsodaName | medium |
---|---|
BclA CBDclos glycerol stock | LB(CP) |
INP-CBDcex glycerol stock | LB(CP) |
INP-CBDclos glycerol stock | LB(CP) |
9/16
Transformation
Uchino, SassaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
8/20 J23105 RFP 1/100 | 20 | DH5α /20 | LB(CP) |
9/19
Transformation
Sassa, TakimotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/24 BAN-cex | 1 | DH5α /20 | LB(CP) |
8/5 INP-cex | 1 | DH5α /20 | LB(CP) |
2/8 INP HIsR | 1 | DH5α /20 | LB(CP) |
2/19 BAN-clos | 1 | DH5α /20 | LB(CP) |
8/5 INP-clos | 1 | DH5α /20 | LB(CP) |
2/4 E1010 | 1 | DH5α /20 | LB(CP) |
8/18 BclA-scFv | 1 | DH5α /20 | LB(CP) |
9/19
Liquid Culture
Sassa, TakimotoName | medium |
---|---|
9/15 Liquid culture BclA scFv 3C5[2] | LB(CP) /20 |
9/19 Liquid culture Bcla CTL | LB(CP) /20 |
9/15 Liquid culture INP-scFv 3C5 | LB(CP) /20 |
9/15 Liquid culture INP CTL | LB(CP) /20 |
9/20
Liquid Culture
SassaName | medium |
---|---|
9/19 liquid culture BclA-scFv 3C5-2 | LB(CP) |
9/19 liquid culture BclA-ctl | LB(CP) |
9/19 liquid culture INP-scFv 3C5 | LB(CP) |
9/19 liquid culture INP-ctl | LB(CP) |
9/20
Western Blotting
Ohara, Sassalane | sample |
---|---|
Marker | M.Marker |
1 | INPNC-6His-scFv 300 µl+VLP3 µl |
2 | INPNC-6His-scFv 300 µl+PBS3 µl |
3 | INPNC-6His-ctl300 µl+VLP3 µl |
4 | BclA-6His-scFv 300 µl+VLP3 µl |
5 | BclA-6His-scFv 300 µl+PBS3 µl |
6 | BclA-6His-ctl 300 µl+VLP3 µl |
7 | VLP0.5 µl |
8 | VLP0.2 µl |
![](https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--9_20_No124_General_Miyazaki_10_17_06_17_54.jpg)
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 80 sec
(ECL Prime)
9/20
Transformation
UchinoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
BclA-clos glycerol stock | - | DH5α /20 | LB(CP) |
INP-clos glycerol stock | - | DH5α /20 | LB(CP) |
9/21
PCR (Steps)
UchinoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 65℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 3min | 1 |
Final Holding | 8℃ | - | - |
9/21
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 miniprep INP-His9 /0.2 | VF /0.75 | m.scFv Rv /0.75 | - | 10.8 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His9 /0.2 | m.scFv Fw /0.75 | VR /0.75 | - | 10.8 | KAPA HiFi Ready Mix /12.5 | 25 |
VF 10µM VR 10µM
9/21
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 100bp ladder | 5 |
3 | 9/21 PCR INP-His9-1 | 25 |
4 | 9/21 PCR INP-His9-2 | 25 |
![](https://static.igem.org/mediawiki/2016/5/53/T--Kyoto--9_21_No129_Gel_Extraction_Notsu_10_03_01_11_36.1.jpg)
![](https://static.igem.org/mediawiki/2016/b/b2/T--Kyoto--9_21_No129_Gel_Extraction_Notsu_10_03_01_11_36.2.jpg)
9/21
Liquid Culture
Ohara, SassaName | medium |
---|---|
9/19 Liquid culture BAN-scFv 3C5[2] | LB(CP) |
9/19 Liquid culture BAN-CTL | LB(CP) |
9/19 Liquid culture INP-scFv 3C5 | LB(CP) |
9/19 Liquid culture INP-CTL | LB(CP) |
9/15 BAN- scFv 1C3 | LB(CP) |
9/15 BAN-CBDcex | LB(CP) |
9/21
Western Blotting
Ohara, Sassalane | sample |
---|---|
Marker | M.Marker |
1 | INPNC-6His-scFv 200 µl+VLP2 µl |
2 | INPNC-6His-scFv 200 µl+PBS2µl(NC) |
3 | INPNC-6His-ctl200 µl+VLP2 µl(NC) |
4 | BclA-6His-scFv200 ul(Vector pSB3C5)+VLP2 ul |
5 | BclA-6His-scFv 200 µl+VLP2 µl (NC) |
6 | BclA-6His-scFv(Vector pSB1C3)+VLP2 ul |
7 | BclA-6His-CBDcex 200 ul + VLP2 ul (NC) |
8 | PBS200 ul +VLP2 ul (NC) |
9 | VLP1 ul (PC) |
![](https://static.igem.org/mediawiki/2016/1/16/T--Kyoto--9_21_No131_General_Miyazaki_10_17_06_47_39.jpg)
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 60 sec
(ECL Prime)
9/21
Gel Extraction(Measurement)
UchinoSample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
---|---|---|---|
9/21 PCR INP-His9-1 | 19.3 | 1.63 | 0.54 |
9/21 PCR INP-His9-2 | 48.6 | 1.82 | 0.24 |
9/21
PCR (Steps)
UchinoSteps | Temparature | Time | Cycle |
---|---|---|---|
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 65℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 3min | 1 |
Final Holding | 8℃ | - | - |
9/21
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
9/21 Gel Extract INP-His9-1 /1µl+9/21 Gel Extract INP-His9-2 /0.5µl | VF /0.75 | VR /0.75 | - | 9.5 | KAPA HiFi /12.5 | 25 |
9/22
growth carve
Sassasample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h |
---|---|---|---|---|---|---|---|---|---|---|---|
BAM scFv [1] | -0.048 | -0.047 | -0.048 | 1.245 | -0.317 | -0.308 | -0.149 | ||||
BAM scFv [2] | -0.036 | 1.512 | -0.830 | -0.309 | -0.314 | -0.127 | |||||
INP scFv [1] | -0.047 | -0.050 | -0.644 | 1.045 | -0.307 | -0.153 | -0.142 | ||||
INP scFv [2] | -0.038 | -0.o25 | -0.186 | -0.280 | 0.000 | -0.289 | |||||
BAN CBDcex [1] | -0.045 | -0.048 | -0.044 | 1.089 | -0.322 | -0.284 | -0.296 | ||||
BAN CBDcex [2] | -0.034 | 1.204 | -1.057 | -0.311 | 0.268 | -0.285 | |||||
BAN CBDclos [1] | 0.035 | -0.037 | -0.035 | 1.261 | -0.323 | -0.160 | -0.287 | ||||
BAN CBDclos [2] | -0.018 | 1.306 | -0.050 | -0.282 | -0.128 | -0.293 | |||||
INP CBDcex [1] | -0.020 | -0.027 | -0.016 | 1.245 | -0.295 | -0.263 | -0.288 | ||||
INP CBDcex [2] | -0.027 | 1.608 | 0.016 | -0.287 | -0.224 | -0.294 | |||||
INP CBDclos [1] | -0.034 | -0.034 | -0.032 | 1.026 | -0.318 | -0.235 | -0.270 | ||||
INP CBDclos [2] | -0.026 | 1.415 | 0.012 | -0.297 | -0.047 | -0.272 | |||||
BAN CTL [1] | -0.039 | -0.039 | -0.036 | 1.274 | -0.314 | -0.230 | -0.271 | ||||
BAN CTL [2] | -0.056 | 1.530 | -0.060 | -0.284 | -0.084 | -3.11 | |||||
INP CTL [1] | -0.042 | -0.043 | -0.042 | 1.134 | -0.235 | -0.181 | -0.296 | ||||
INP CTL [2] | -0.042 | -1.488 | -0.030 | -0.288 | -0.109 | -0.295 | |||||
E1010 [1] | -0.041 | -0.040 | -0.036 | 1.171 | -0.322 | -0.252 | -0.271 | ||||
E1010 [2] | -0.030 | 1.563 | -0.061 | -0.267 | 0.286 | -0.267 | |||||
blank [1]/[2] | /-0.307 | -0.306/-0.305 | -0.307/-0.271 | 0.306/ |
9/22
Liquid Culture
Sassa, MiyazakiName | medium |
---|---|
9/21 liquid culture BAN-CBDcex | LB(AMP) |
9/15 liquid culture INP-ctl | LB(AMP) |
9/15 liquid culture INP-CBDcex | LB(AMP) |
9/15 liquid culture INP-CBDclos | LB(AMP) |
9/15 liquid culture BAN-scFv 3C5 | LB(AMP) |
9/15 liquid culture INP-scFv 3C5 | LB(AMP) |
9/19 liquid culture BAN-CBDclos | LB(AMP) |
9/15 liquid culture BAN-ctl | LB(AMP) |
9/16 transformation J23105-RFP | LB(AMP) |
9/23
SEM
Miyazakinumber | sample(µl) | VLP(µl) | PBS | centrifugal force |
---|---|---|---|---|
1 | INP scFv /25 | 2.5 | 0 | ×3000g |
2 | INP CTL /25 | 2.5 | 0 | ×3000g |
3 | BclA scFv /25 | 2.5 | 0 | ×3000g |
4 | BclA CTL /25 | 2.5 | 0 | ×3000g |
5 | BclA scFv /25 | 0 | 2.5 | ×3000g |
6 | INP scFv /25 | 2.5 | 0 | ×200g |
7 | INP CTL /25 | 2.5 | 0 | ×200g |
8 | BclA scFv /25 | 2.5 | 0 | ×200g |
9 | BclA CTL /25 | 2.5 | 0 | ×200g |
10 | BclA scFv /25 | 0 | 2.5 | ×200g |
11 | 0 | 2.5 | 0 | ×200g |
9/23
RNA extraction
Isodanumber | sample name |
---|---|
1 | BclA-CBD cex |
2 | BclA-CBDclos |
3 | INP-CBDcex |
4 | INP-CBDclos |
5 | BclA-scFv |
6 | INP-scFv |
9/23
growth carve
Sassasample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h | 9h | 10h |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
9/19 Liquid culture BAN scFv[1] | 0.003 | 0.001 | 0.000 | 0.008 | 0.015 | 0.009 | 0.013 | 0.016 | 0.015 | 0.022 | 0.062 | 0.020 | 0.027 |
9/19 Liquid culture BAN scFv[2] | 0.013 | 0.012 | 0.000 | 0.000 | 0.000 | 0.000 | 0.001 | 0.011 | 0.011 | 0.015 | 0.022 | 0.027 | 0.057 |
9/19 Liquid culture INP scFv[1] | 0.012 | 0.000 | 0.000 | 0.011 | 0.006 | 0.006 | 0.007 | 0.008 | 0.013 | 0.025 | 0.036 | 0.098 | 0.076 |
9/19 Liquid culture INP scFv[2] | 0.007 | 0.005 | 0.000 | 0.002 | 0.003 | 0.002 | 0.012 | 0.012 | 0.019 | 0.029 | 0.034 | 0.047 | 0.072 |
9/19 Liquid culture BAN cex[1] | 0.014 | 0.005 | 0.010 | 0.008 | 0.011 | 0.009 | 0.014 | 0.014 | 0.016 | 0.029 | 0.038 | 0.043 | 0.083 |
9/19 Liquid culture BAN cex[2] | 0.011 | 0.011 | 0.010 | 0.008 | 0.004 | 0.004 | 0.020 | 0.021 | 0.036 | 0.047 | 0.065 | 0.087 | 0.109 |
9/19 Liquid culture BAN clos[1] | 0.000 | 0.000 | 0.000 | 0.002 | 0.009 | 0.003 | 0.009 | 0.014 | 0.019 | 0.014 | 0.016 | 0.024 | 0.046 |
9/19 Liquid culture BAN clos[2] | 0.005 | 0.007 | 0.000 | 0.000 | 0.000 | 0.003 | 0.012 | 0.022 | 0.020 | 0.035 | 0.055 | 0.086 | 0.117 |
9/19 Liquid culture INP cex[1] | 0.187 | 0.000 | 0.001 | 0.007 | 0.011 | 0.006 | 0.013 | 0.016 | 0.020 | 0.023 | 0.037 | 0.048 | 0.068 |
9/19 Liquid culture INP cex[2] | 0.002 | 0.009 | 0.000 | 0.001 | 0.001 | 0.002 | 0.015 | 0.017 | 0.023 | 0.036 | 0.053 | 0.080 | 0.111 |
9/19 Liquid culture INP clos[1] | 0.006 | 0.000 | 0.000 | 0.023 | 0.011 | 0.003 | 0.011 | 0.013 | 0.016 | 0.022 | 0.033 | 0.058 | 0.068 |
9/19 Liquid culture INP clos[2] | 0.029 | 0.028 | 0.000 | 0.009 | 0.000 | 0.016 | 0.001 | 0.007 | 0.008 | 0.014 | 0.020 | 0.029 | 0.074 |
9/19 Liquid culture BAN CTL[1] | 0.004 | 0.000 | 0.000 | 0.000 | 0.002 | 0.000 | 0.002 | 0.012 | 0.015 | 0.012 | 0.027 | 0.055 | 0.081 |
9/19 Liquid culture BAN CTL[2] | 0.002 | 0.002 | 0.001 | 0.000 | 0.001 | 0.005 | 0.017 | 0.024 | 0.031 | 0.042 | 0.059 | 0.073 | 0.113 |
9/19 Liquid culture INP CTL[1] | 0.011 | 0.000 | 0.032 | 0.013 | 0.017 | 0.010 | 0.015 | 0.033 | 0.031 | 0.035 | 0.037 | 0.019 | 0.026 |
9/19 Liquid culture INP CTL[2] | 0.013 | 0.020 | 0.000 | 0.001 | 0.000 | 0.003 | 0.009 | 0.016 | 0.021 | 0.026 | 0.044 | 0.059 | 0.136 |
9/19 Liquid culture E1010[1] | 0.015 | 0.016 | 0.014 | 0.011 | 0.016 | 0.018 | 0.032 | 0.066 | 0.108 | 0.173 | 0.258 | 0.361 | 0.393 |
9/19 Liquid culture E1010[2] | 0.064 | 0.010 | 0.007 | 0.011 | 0.012 | 0.019 | 0.029 | 0.041 | 0.061 | 0.097 | 0.150 | 0.180 | - |
9/23
OEPCR
Uchinosample1 /µl | sample2 /µl | primer Fw /µl | primer Rv /µl | milliQ /µl | KAPA HiFi /µl | total |
---|---|---|---|---|---|---|
9/21 Gel Extract1 /1 | 9/21 Gel Extract2 /0.5 | VF/ 0.75 | VR /0.75 | 9.5 | 12.5 | 25 |
9/23
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/3 | |
2 | 9/23 OEPCR1 /25 | |
3 | 9/23 OEPCR2 /25 |
![](https://static.igem.org/mediawiki/2016/8/82/T--Kyoto--9_23_No141_Electrophoresis_Miyazaki_10_06_03_34_18.jpg)
9/23
PCR (Target)
YamamotoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 miniprep INP-His-scFv9 /0.4 | m.scFv Fw/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | m.scFv Fw/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | VF/1.25 | m.scFv Rv/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | VF/1.25 | m.scFv Rv/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
9/23 Gel Extraction INP-scFv m.PCR/1.5 | VF/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 8.5 | 0 | 25 |
9/26
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/4 | |
2 | 9/23PCR1 /25 | |
3 | 9/23PCR2 /25 | |
4 | 9/23PCR3 /25 | |
5 | 9/23PCR4 /25 | |
6 | 9/23PCR5 /25 |
![](https://static.igem.org/mediawiki/2016/6/68/T--Kyoto--9_26_No143_Electrophoresis_Miyazaki_10_06_03_46_00.1.jpg)
![](https://static.igem.org/mediawiki/2016/1/18/T--Kyoto--9_26_No143_Electrophoresis_Miyazaki_10_06_03_46_00.2.jpg)
9/26
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
9/26 electrophoresis lane 4,5 | 0.357g | |
9/26 electrophoresis lane 2,3 | 0.354g |
9/26
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
9/26 Gel Extract lane4,5 /2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 7 | 25 | |
9/26 Gel Extract lane2,3 /2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 7 | 25 |
9/26
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
Bcla-cex 8/5 miniprep | BL21 | LB(CP) | |
Bcla-CTL 8/4 miniprep | BL21 | LB(CP) | |
Bcla-cex 8/5 miniprep | BL21 | LB(CP) | |
Bcla-CTL 8/4 miniprep | BL21 | LB(CP) |
9/26
RNA extraction
Isodanumber | sample | conc.µg/µl | 260/280 | 260/230 |
---|---|---|---|---|
4 | BclAcex | 74.73 | 1.94 | 1.61 |
2 | INPcex | 83 | 1.97 | 1.38 |
5 | INPclos | 120.21 | 1.99 | 1.94 |
3 | INPclos | 58.23 | 1.98 | 1.84 |
6 | BclAscFV | 74.76 | 1.96 | 1.84 |
7 | INPscFv | 80.6 | 1.97 | 1.97 |
9/27
growth carve
Sassa, Takimotosample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h |
---|---|---|---|---|---|---|---|---|---|---|---|
9/21Liquid culture BAN scFv | 0.113 | 0.170 | 0.266 | 0.408 | 0.599 | 0.893 | 0.164(1.64) | 0.169(1.69) | 0.232(2.32) | 0.234(2.34) | 0.221(2.21) |
9/22Liquid culture BAN cex | 0.109 | 0.147 | 0.219 | 0.374 | 0.515 | 0.873 | 0.146(1.46) | 0.160(1.60) | 0.176(1.76) | 0.231(2.31) | 0.260(2.60) |
9/19Liquid culture BAN clos | 0.097 | 0.109 | 0.184 | 0.338 | 0.486 | 0.744 | 0.130(1.30) | 0.173(1.73) | 0.199(1.99) | 0.203(2.03) | 0.274(2.74) |
9/21Liquid culture BAN CTL | 0.106 | 0.170 | 0.252 | 0.304 | 0.553 | 0.797 | 0.177(1.77) | 0.155(1.55) | 0.216(2.16) | 0.236(2.36) | 0.257(2.57) |
9/21Liquid culture INP scFv | 0.088 | 0.083 | 0.101 | 0.122 | 0.299 | 0.409 | 0.073(0.73) | 0.121(1.21) | 0.140(1.40) | 0.143(1.43) | 0.195(1.95) |
9/15Liquid culture INP cex | 0.102 | 0.116 | 0.145 | 0.207 | 0.320 | 0.626 | 0.170(1.70) | 0.143(1.43) | 0.176(1.76) | 0.188(1.88) | 0.238(2.38) |
9/22Liquid culture INP clos | --- | 0.133 | 0.209 | 0.373 | 0.572 | 0.930 | 0.156(1.56) | 0.201(2.01) | 0.246(2.46) | 0.237(2.37) | 0.293(2.93) |
9/21Liquid culture INP CTL | 0.112 | 0.155 | 0.237 | 0.409 | 0.582 | 0.955 | 0.153(1.53) | 0.209(2.09) | 0.268(2.68) | 0.288(2.88) | 0.276(2.76) |
9/21Liquid culture E1010 | 0.073 | 0.091 | 0.149 | 0.265 | 0.396 | 0.706 | 0.157(1.57) | 0.182(1.82) | 0.222(2.22) | 0.222(2.22) | 0.246(2.46) |
rpm 160
total10mL
CP 2mL/1L
measured 10 fold dilution after 4h
9/27
Western Blotting
Oharalane | sample |
---|---|
Marker | M.Marker(Wako) |
1 | BclA6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) |
2 | BclA6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
3 | INPNC6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) |
4 | INPNC6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
5 | BclA6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) |
6 | BclA6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) |
7 | INPNC6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) |
8 | INPNC6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) |
9 | PBS 300µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
![](https://static.igem.org/mediawiki/2016/3/3d/T--Kyoto--9_27_No149_General_Miyazaki_10_17_06_53_43.png)
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 60 sec
(ECL Prime)
9/27
Western Blotting
Ohara, Sassalane | sample |
---|---|
Marker | M.Marker(Wako) 5µl |
1 | BBa_E1010(NC) |
2 | BclA-6His-CBDclos (17 kDa) |
3 | INPNC-6His-CBDclos(47 kDa) |
4 | BclA-6His-CBDcex (17 kDa) |
5 | INPNC-6His-CBDcex (47 kDa) |
6 | BclA-6His-scFv (33 kDa) |
7 | INPNC-6His-scFv (63 kDa) |
8 | BBa_K875004 (PC) Lpp-OmpA-scFv-6His (43kDa) |
Marker | M.Marker(Wako) 3µl |
9 | BBa_K875004 (PC) Lpp-OmpA-scFv-6His(43 kDa) |
![](https://static.igem.org/mediawiki/2016/2/2a/T--Kyoto--9_27_No149_General_Miyazaki_10_17_07_01_58.png)
Used membrane fraction from 100ml culture at logarithmic growth phase.
Transfer ;60min 100mA(CC)
Blocking ;1h
1’antibody ;anti6His(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with TBST 1h
Detection time 380sec
(ECL Prime)
9/27
Reverse Transcription
Yamadanumber | Template/µl | gDNA wipeout buffer/µl | RNAase free water/µl | total/µl |
---|---|---|---|---|
A1 | BclAcex/12 | 2 | 0 | 14 |
A2 | BclAclos/12 | 2 | 0 | 14 |
A3 | INPcex/8.32 | 2 | 3.68 | 14 |
A4 | INPclos/12 | 2 | 0 | 14 |
A5 | BclAscFv/12 | 2 | 0 | 14 |
A6 | INPscFv/12 | 2 | 0 | 14 |
B1 | BclAcex/12 | 2 | 0 | 14 |
B2 | BclAclos/12 | 2 | 0 | 14 |
B3 | INPcex/8.32 | 2 | 3.68 | 14 |
B4 | INPclos/12 | 2 | 0 | 14 |
B5 | BclAscFv/12 | 2 | 0 | 14 |
B6 | INPscFv/12 | 2 | 0 | 14 |
9/27
Reverse Transcription
Yamadanumber | template/µl | RTase/µl | RNAase free water/µl | RT buffer/µl | Primer Mix/µl | Total |
---|---|---|---|---|---|---|
A1 | BclAcex/14 | 1 | 0 | 4 | 1 | 20 |
A2 | BclAclos/14 | 1 | 0 | 4 | 1 | 20 |
A3 | INPcex/14 | 1 | 0 | 4 | 1 | 20 |
A4 | INPclos/14 | 1 | 0 | 4 | 1 | 20 |
A5 | BclAscFv/14 | 1 | 0 | 4 | 1 | 20 |
A6 | INPscFv/12 | 1 | 0 | 4 | 1 | 20 |
B1 | BclAcex/14 | 0 | 1 | 4 | 1 | 20 |
B2 | BclAclos/14 | 0 | 1 | 4 | 1 | 20 |
B3 | INPcex/14 | 0 | 1 | 4 | 1 | 20 |
B4 | INPclos/14 | 0 | 1 | 4 | 1 | 20 |
B5 | BclAscFv/14 | 0 | 1 | 4 | 1 | 20 |
B6 | INPscFv/12 | 0 | 1 | 4 | 1 | 20 |
9/27
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/5 | |
2 | 9/26 PCR INP- His scFv/25 | |
3 | 9/26 PCR INP- His scFv/25 |
![](https://static.igem.org/mediawiki/2016/e/ed/T--Kyoto--9_27_No152_Electrophoresis_Miyazaki_10_06_04_10_12.1.jpg)
![](https://static.igem.org/mediawiki/2016/a/a0/T--Kyoto--9_27_No152_Electrophoresis_Miyazaki_10_06_04_10_12.2.jpg)
9/27
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
- | 9/27 Electrophoresis INP-His-scFv | 0.7660g |
nanodrop
17.8ng/nl
260/280 1.79
260/230 0.31
9/27
CBD 15th
Yamadasample | before | after | wash |
---|---|---|---|
BclAcex+ (CB buffer) | 0.181/0.212/0.204 | 0.254/0.255/0.254 | 0/0/0 |
BclAcex- (CB buffer) | 0.208/0.275/0.228 | 0.160/0.171/0.162 | |
BclActl+ (CB buffer) | 0.178/0.176/0.171 | 0.082/0.085/0.082 | 0.022/0.02/0.02 |
BclActl- (CB buffer) | 0.218/0.214/0.216 | 0.161/0.160/0.159 | |
BclAcex+ (PBS buffer) | 0.212/0.211/0.210 | 0.333/0.334/0.333 | 0.022/0.022/0.021 |
BclAcex- (PBS buffer) | 0.203/0.202/0.201 | 0.178/0.178/0.180 | |
BclActl+ (PBS buffer) | 0.182/0.181/0.180 | 0.253/0.246/0.246 | 0.021/0.021/0.021 |
BclActl- (PBS buffer) | 0.208/0.205/0.208 | 0.192/0.194/0.194 |
9/29
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
9/27 Gel Extract/2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 9 | 25 |
9/29
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder/2 | |
2 | 9/29 PCR INP-His-scFv/2 | |
3 | 9/29 PCR INP-His-scFv/2 |
![](https://static.igem.org/mediawiki/2016/3/3e/T--Kyoto--9_29_No157_Electrophoresis_Miyazaki_10_06_04_22_37.jpg)
9/29
PCR (Purification)
UchinoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
9/29 PCR INP-His-scFv | 31.9 | 2.02 | 1.38 |
9/30
Electrophoresis
Uchino, MiyazakiLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder | |
2 | BclA clos/4.0 | |
3 | INP clos/5.5 | |
4 | BclA cex/4.5 | |
5 | INP cex/2.5 | |
6 | BclA scFv/4.5 | |
7 | INP scFv/4.0 |
10/1
RNA purification
Oharasample | OD |
---|---|
BclAcex | 0.504 |
BclAscFv | 0.788 |
INPscFv | 0.837 |
INPclos | 1.564 |
BclAclos | 0.817 |
INPcex | 0.839 |
![](https://static.igem.org/mediawiki/2016/d/d1/T--Kyoto--10_1_No161_General_Miyazaki_10_11_10_14_22.jpg)
10/1
RNA extraction
Uchinonumber | sample | conc(ng/µl) | 260/280 | 260/230 | diluted solution volume(200ng/µl) |
---|---|---|---|---|---|
1 | INPclos | 359.2 | 2.07 | 1.21 | 11.94 |
2 | BclAcex | 230.9 | 2.03 | 0.85 | 2.31 |
3 | INPcex | 1069.0 | 2.11 | 1.43 | 65.17 |
4 | INPscFv | 320.0 | 2.10 | 0.93 | 9 |
5 | BclAclos | 386.2 | 2.04 | 0.97 | 13.97 |
6 | BclAcex | 441.0 | 2.04 | 0.83 | 18 |
10/1
Reverse Transcription
Uchinoname | copy(high or low) | RT |
---|---|---|
3 nega | high copy | - |
3 posi | high copy | + |
4 nega | low copy | - |
4 posi | low copy | + |
10/1
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
10/1 RT×20 3RT- /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
10/1 RT×20 3RT+ /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
10/1 RT×20 4RT- /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
10/1 RT×20 4RT+ /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
9/27 RT×1 A-6 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
9/27 RT×1 A-5 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
9/27 RT×1 B-6 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl | |
9/27 RT×1 B-5 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | 10µl |
10/1
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
10/1 RT×20 3RT- /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
10/1 RT×20 3RT+ /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
10/1 RT×20 4RT- /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
10/1 RT×20 4RT+ /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
9/27 RT×1 A-6 /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
9/27 RT×1 A-5 /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
9/27 RT×1 B-6 /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 | |
9/27 RT×1 B-5 /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | 10 |
10/1
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 3RT- target /10 | |
2 | 3RT+ target /10 | |
3 | 3RT- 16s rRNA /10 | |
4 | 3RT+ 16s rRNA /10 | |
5 | 4RT- target /10 | |
6 | 4RT+ target /10 | |
7 | 4RT- 16s rRNA /10 | |
8 | 4RT+ 16s rRNA /10 | |
9 | 100bp ladder /5 | |
10 | A-6 target /10 | |
11 | A-5 target /10 | |
12 | A-6 16s rRNA /10 | |
13 | A-5 16s rRNA /10 | |
14 | B-6 target /10 | |
15 | B-5 target /10 | |
16 | B-6 16s rRNA /10 | |
17 | B-5 16s rRNA /10 |
![](https://static.igem.org/mediawiki/2016/4/4a/T--Kyoto--10_1_No165_Electrophoresis_Miyazaki_10_10_07_19_39.jpg)
10/1
Transformation
OharaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
K165002 | DH5α | ||
BclA scFv | DH5α | ||
INP scFv | DH5α | ||
BclA cex | DH5α | ||
INP cex | DH5α | ||
BclA clos | DH5α | ||
INP clos | DH5α | ||
K165002 | BL21 | ||
BclA scFv | BL21 | ||
INP scFv | BL21 | ||
BclA cex | BL21 | ||
INP cex | BL21 | ||
BclA clos | BL21 | ||
INP clos | BL21 |
10/4
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
INP CTL 8/4 miniprep | DH5α/20 | LB(CP) |
10/5
Western Blotting
Oharalane | sample |
---|---|
Marker | M.Marker(Wako) 5µl |
1 | BBa_E165002(NC) |
2 | BclA-6His-scFv (33 kDa) |
3 | INPNC-6His-scFv (63 kDa) |
4 | BclA-6His-CBDcex (17 kDa) |
5 | INPNC-6His-CBDcex(47kDa) |
6 | BclA-6His-CBDclos (17 kDa) |
7 | INPNC-6His-CBDclos (47 kDa) |
8 | BBa_K875004(PC) Lpp-OmpA-scFv-6His (43 kDa) |
![](https://static.igem.org/mediawiki/2016/c/cb/T--Kyoto--10_5_No193_General_Miyazaki_10_17_07_20_04.png)
Used pelet from 400 µl overnight culture.
Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP 1:2500 with TBST 1h
Detection time 240 sec
(ECL Prime)
10/6
Liquid Culture
UchinoName | medium |
---|---|
10/5 liquid culture INP CTL /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CTL /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CBD cex(5) /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CBD cex(5) /2.5ml | LB(CP) /47.5ml |
10/6
Liquid Culture
YamadaName | medium |
---|---|
10/5 liquid culture INP CTL /100ml | LB(CP) /100ml |
10/3 liquid culture INP CBD cex /100ml | LB(CP) /200ml |
10/7
CBD 16th
Yamadasample | before | after | wash |
---|---|---|---|
INPcex+ | 0.208/0.208/0.211 | 0.224/0.221/0.226 | 0.013/0.012/0.010 |
INPcex- | 0.210/0.216/0.204 | 0.232/0.231/0.232 | |
INPctl+ | 0.196/0.196/0.191 | 0.220/0.217/0.212 | 0.023/0.024/0.025 |
INPctl- | 0.194/0.195/0.196 | 0.207/0.199/0.208 | |
cellulose only | 0.015/0.013/0.015 | 0.031/0.031/0.032 | 0.018/0.017/0.019 |
10/11
Transformation
YamadaSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
2/25 miniprep INPcex | 3 | DH5α /20 | LB(CP)×3 |
2/8 miniprep INP His 12 (INPscFv) | 3 | DH5α /20 | LB(CP)×3 |
2/25 miniprep INPcex | 3 | BL21 /20 | LB(CP)×3 |
2/8 miniprep INP His 12 (INPscFv) | 3 | BL21 /20 | LB(CP)×3 |
10/11
SEM(CBD)
Miyazakisample/ml | before | after | wash1 | wash2 |
---|---|---|---|---|
INP CBDcex 10/1 liquid culture /90 | 0.550 | 0.392 | 0.074/0.084 | -0.004/-0.018 |
INP CBDcex 10/1 liquid culture /45 | 0.550 | 0.432 | 0.002/0.000 | -0.001/0.000 |
10/12
Colony PCR (typeA)
YamadaTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INP-His-scFv-INP-His-CBDcex DH5α colony1 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony2 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony3 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony4 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony6 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony7 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony8 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony9 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony10 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony11 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony12 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony13 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony14 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony15 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony16 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony17 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony18 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony19 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony20 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
10/12
Colony PCR (typeA)
YamadaTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
2/8 miniprep INP-His-scFv /0.5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
2/25 miniprep INP-His-CBDcex /0.5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
10/12
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony1 /5 | |
2 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony2 /5 | |
3 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony3 /5 | |
4 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony4 /5 | |
5 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony5 /5 | |
6 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony6 /5 | |
7 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony7 /5 | |
8 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony8 /5 | |
9 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony17 /5 | |
10 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony18 /5 | |
11 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony19 /5 | |
12 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony20 /5 | |
13 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony21 /5 | |
14 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony22 /5 | |
15 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony23 /5 | |
16 | INPNC-His-CBDcex 10/12 colony PCR /5 | |
17 | 1kbp ladder /10 |
![](https://static.igem.org/mediawiki/2016/1/11/T--Kyoto--10_12_No172_Electrophoresis_Miyazaki_10_15_05_41_02.jpg)
10/12
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 INP-His 12 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/13
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/8 INP-His 12 /0.1 | VF /0.75 | VR /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/13
Colony PCR
Yamadasample /µl | primer /µl | KAPA Taq Extra Hotstart /µl | DMSO /µl | MilliQ /µl | total /µl |
---|---|---|---|---|---|
INP-cex + INP-scFv colony2 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony7 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony17 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony19 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony9 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony10 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony11 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony12 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony13 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony16 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony23 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony24 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony26 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony27 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony28 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony29 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony30 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony31 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
10/13
Colony PCR
Yamadasample /µl | primer /µl | KAPA Taq Extra Hotstart /µl | DMSO /µl | MilliQ /µl | total /µl |
---|---|---|---|---|---|
INP-cex + INP-scFv colony31 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony32 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony33 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony34 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-scFv /0.25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex /0.25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
10/13
Miniprep
UchinoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
10/6 liquid culture INP-His-CBDcex | 27.8 | 1.96 | 1.86 |
10/13
PCR (Purification)
UchinoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
10/12 PCR INP-His 12 (INP-His coding) | 727.3 | 1.88 | 1.86 |
10/13 PCR INP-His 12 | 1132.1 | 1.89 | 2.04 |
10/13
Restriction Enzyme Digestion
UchinoSample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
10/13 PCR purification INP-His coding | 7 | 2 | 0 | 0 | 2 | 10×H /6 | 0 | 44 | 60 |
10/13 PCR purification INP-His 12 | 6 | 0 | 2 | 0 | 2 | 10×M /6 | 0 | 44 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 10×H /6 | 0 | 0 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 0 | 0 | 2 | 2 | 10×H /6 | 0 | 0 | 60 |
10/13
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
1 | INPcex+INPscFv colony2 | |
2 | INPcex+INPscFv colony7 | |
3 | INPcex+INPscFv colony19 | |
4 | INPcex+INPscFv colony9 | |
5 | INPcex+INPscFv colony17 | |
6 | INPcex+INPscFv colony10 | |
7 | INPcex+INPscFv colony11 | |
8 | INPcex+INPscFv colony12 | |
9 | INPcex+INPscFv colony15 | |
10 | INPcex+INPscFv colony16 | |
11 | INPcex+INPscFv colony23 | |
12 | INPcex+INPscFv colony24 | |
13 | INPcex+INPscFv colony25 | |
14 | INPcex+INPscFv colony26 | |
15 | INPscFv /5 | |
16 | INPcex /5 | |
17 | 1kbp ladder |
![](https://static.igem.org/mediawiki/2016/1/15/T--Kyoto--10_13_No178_Electrophoresis_Miyazaki_10_16_03_04_04.jpg)
10/13
Electrophoresis
YamadaLane | sample/(µl) | Length(bp) |
---|---|---|
A1 | INPcex+INPscFv colony2 | |
A2 | INPcex+INPscFv colony7 | |
A3 | INPcex+INPscFv colony19 | |
A4 | INPcex+INPscFv colony9 | |
A5 | INPcex+INPscFv colony17 | |
A6 | INPscFv /5 | |
A7 | INPcex /5 | |
A8 | 1kbp ladder /5 | |
B1 | INPcex+INPscFv colony10 | |
B2 | INPcex+INPscFv colony11 | |
B3 | INPcex+INPscFv colony12 | |
B4 | INPcex+INPscFv colony13 | |
B5 | INPcex+INPscFv colony14 | |
B6 | 1kbp ladder /5 |
![](https://static.igem.org/mediawiki/2016/2/26/T--Kyoto--10_13_No179_Electrophoresis_Miyazaki_10_16_03_20_40.jpg)
10/13
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kbp ladder | |
2 | 10/13 1A | |
3 | 10/13 2A | |
4 | 10/13 3A | |
5 | 10/13 4A | |
6 | 10/13 5A | |
7 | 10/13 1B | |
8 | 10/13 2B | |
9 | 10/13 3B | |
10 | 10/13 4B | |
11 | 10/15 5B |
![](https://static.igem.org/mediawiki/2016/e/e2/T--Kyoto--10_13_No180_Electrophoresis_Miyazaki_10_16_03_25_22.jpg)
10/13
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
1A+2A | 10/13 PCR purification INP His coding | |
3A~5A | 10/13 midiprep INP His CBDcex | |
1B+2B | 10/13 PCR purification INP His12 | |
3B~5B | 10/13 midiprep INP His CBDcex |
![](https://static.igem.org/mediawiki/2016/0/05/T--Kyoto--10_13_No181_Gel_Extraction_Miyazaki_10_16_03_33_03.jpg)
10/13
Fluorescence Microscopy
Uchinosample | number | magnification |
---|---|---|
INPclos | 13318 | ×200 |
INPclos | 13319~13328 | ×600 |
INP CTL | 13329 | ×200 |
INP CTL | 13330~13338 | ×600 |
cellulose only | 13339 | ×200 |
cellulose only | 13340,13343~13351 | ×600 |
cellulose only | 13341,13342 | ×1000 |
without filter
10/13
Fluorescence Microscopy
UchinoE.coli | OD600 | cellulose | weight | filter |
---|---|---|---|---|
- | - | powder | 10 | non-filtered |
INPNC-His-ctl | 0.2 | powder | 10 | non-filtered |
INPNC-His-CBDcex | 0.2 | powder | 10 | non-filtered |
- | - | powder | 10 | filtered |
INPNC-His-ctl | 0.2 | powder | 10 | filtered |
INPNC-His-CBDcex | 0.2 | powder | 10 | filtered |
10/13
Fluorescence Microscopy
Uchinosample | number | magnification |
---|---|---|
INPcex | 13353 | ×200 |
INPcex | 13354~13356,13364,13366 | ×600 |
INPcex | 13357,13365 | ×1000 |
INP CTL | 13367 | ×200 |
INP CTL | 13368~13378 | ×600 |
INP CTL | 13379 | ×1000 |
cellulose only | 13380,13381 | ×200 |
cellulose only | 13382~13391 | ×600 |
with filter
10/13
Fluorescence Microscopy
Uchinosample | number | magnification |
---|---|---|
INPcex | 13392~98 | ×200 |
INPcex | 13399~13408 | ×600 |
INP CTL | 13309~13419 | ×200 |
bemliese | 13420~13430 | ×200 |
with bemliese
10/14
Electrophoresis
YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kbp ladder /3 | |
2 | 10/13 1A /25 | |
4 | 10/13 2A /25 | |
5 | 10/13 3A /25 | |
6 | 10/13 4A /25 | |
7 | 10/13 5A /25 | |
8 | 10/13 1B /25 | |
9 | 10/13 2B /25 | |
10 | 10/13 3B /25 | |
11 | 10/13 4B /25 | |
12 | 10/13 5B /25 |
![](https://static.igem.org/mediawiki/2016/b/b9/T--Kyoto--10_14_No186_Electrophoresis_Miyazaki_10_16_04_07_57.1.jpg)
![](https://static.igem.org/mediawiki/2016/8/8a/T--Kyoto--10_14_No186_Electrophoresis_Miyazaki_10_16_04_07_57.2.jpg)
with bemliese
10/14
SEM(CBD)
Miyazakisample | membrane shape |
---|---|
INPcex + cellulose | square |
INP CTL + cellulose | triangle |
cellulose only | pentagon |
10/14
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
10/14 electrophoresis 1B longer + 2B longer e1 | 10/13 restriction enzyme design INP His12 | |
10/14 electrophoresis 1B longer + 2B longer e2 | 10/13 restriction enzyme design INP His12 |
10/14
Gel Extraction
UchinoLane | Restriction Enzyme Digestion Product | Volume/(µl) |
---|---|---|
10/14 electrophoresis 1B + 2B | 10/13 restriction enzyme design INP His12 | |
10/14 electrophoresis 1A + 2A | 10/13 restriction enzyme design INP His coding | |
10/14 electrophoresis 3A + 4A + 5A | INP His CBDcex |
10/14
Ligation
Uchinosample1 1A + 2A /µl | sample2 3A~5A /µl | Ligation high ver.2 | total |
---|---|---|---|
5 | 2.5 | 7.5 | 15 |
3.75 | 3.75 | 7.5 | 15 |
10/14
Transformation
UchinoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
INP His coding ligationA | 2 | DH5α /20 | LB(CP) |
INP His coding ligationA | 4 | DH5α /20 | LB(CP) |
INP His coding ligationB | 2 | DH5α /20 | LB(CP) |
INP His coding ligationB | 4 | DH5α /20 | LB(CP) |
10/15
PCR (Purification)
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
10/15 INP-His coding i | 207.4 | 1.87 | 2.11 |
10/15 INP-His coding G | 174.7 | 1.88 | 1.09 |
10/15 INP-His coding E | 175.5 | 1.08 | 2.10 |
10/15 INP-His coding M | 212.9 | 1.88 | 2.14 |
10/15
CBD Binding Assay
Oharasample | Buffer | OD |
---|---|---|
INP cex | CBC buffer | 1.002 |
simultaneous expression | CBC buffer | 0.994 |
INP CTL | CBC buffer | 1.004 |
INP CTL | PBS | 0.964 |
simultaneous expression | PBS | 1.048 |
10/15
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | 10xH Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
---|---|---|---|---|---|---|---|---|---|
10/15 PCR purification INP His coding i | 29.0 | 2 | 0 | 0 | 2 | 6 | 21 | 60 | |
10/15 PCR purification INP His coding G | 34.3 | 2 | 0 | 0 | 2 | 6 | 15.7 | 60 | |
10/15 PCR purification INP His coding E | 34.2 | 2 | 0 | 0 | 2 | 6 | 15.8 | 60 | |
10/15 PCR purification INP His coding M | 28.2(6000ng) | 2 | 0 | 0 | 2 | 6 | 21.8 | 60 | |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | 0 | 60 | |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | 0 | 60 | |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | 0 | 60 | |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | 0 | 60 |
1.5 Dr.Kitabatake
2.6 Agarose S + Promega Gel Extraction
3.7 Agarose S + Dr.Nasuda
4.8 stock
10/15
PCR (Target)
UchinoTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/15
Colony PCR (typeA)
YamamotoTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
A1 10/14 INPcex + INPscFv colony1 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A2 10/14 INPcex + INPscFv colony2 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A3 10/14 INPcex + INPscFv colony3 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A4 10/14 INPcex + INPscFv colony4 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
10/15
Western Blotting
Oharalane | sample |
---|---|
Marker | M.Marker |
1 | BBa_K875004(PC) Lpp-OmpA-scFv-6His 43kDa |
2 | INPNC-6His-CBDcex 47kDa |
3 | INPNC-6His-scFv 63kDa |
4 | INPNC-6His-CBDcex/INPNC-6His-scFv |
5 | BBa_K165002(NC) |
![](https://static.igem.org/mediawiki/2016/0/03/T--Kyoto--10_15_No194_General_Miyazaki_10_17_08_24_33.png)
Used pelet from 500 µl overnight culture, adjested OD600 to 3.1 .
Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP 1:2500 with TBST 1h
Detection time 900 sec
(ECL Prime)
10/16
Electrophoresis
YamamotoLane | sample/(µl) | Length(bp) |
---|---|---|
1kb ladder | ||
10/15 colony PCR colony 1 | 3 | |
10/15 colony PCR colony 2 | 25 | |
10/15 colony PCR colony 3 | 25 | |
10/15 colony PCR colony 4 | 25 |
10/16
Cellulose binding Assay
UchinoCellulose/(mg) | E.coli/(mg) | buffer |
---|---|---|
1 | cex/1 | carbonate-bicarbonate buffer |
1 | ctl/1 | carbonate-bicarbonate buffer |
0.5 | cex/1 | carbonate-bicarbonate buffer |
0.5 | ctl/1 | carbonate-bicarbonate buffer |
0.5 | CBC/1 | carbonate-bicarbonate buffer |
0.1 | cex/1 | carbonate-bicarbonate buffer |
0.1 | ctl/1 | carbonate-bicarbonate buffer |
RT,-3h 500rpm 15:45~21:15 binding
fix with 4×1/100 PFA 10min→centrifuge→PBS→centrifuge
→PBS→centrifuge→PBS→centrifuge→DAP1→centrifuge
→PBS→centrifuge→PBS→through filter→PBS into filter
→centrifuge→PBSinto filter→centrifuge
→suspend with PBS→centrifuge
→suspend with?discoloration inhibitor
10/16
Dual Display Assay for Electron Microscope
E.coli | Cellulose | VLP | buffer |
---|---|---|---|
A.Dual 1mL | 1mg | 10µL | PBS |
B.Dual 1mL | 1mg | PBS | |
C.Dual 1mL | 10µL | PBS | |
D.Dual 1mL | PBS | ||
E.INPctl | 1mg | 10µL | PBS |
F.INPctl | 1mg | PBS | |
G.INPctl | 10µL | PBS | |
H.INPctl | PBS | ||
I | 1mg | PBS | |
J | 10µL | PBS |
RT,ih incubate,850rpm
Dul=1.020
ctl=1.056
10/16
Electrophoresis
Lane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1 kb ladder | 5 |
2 | 10/15 Digestion INP His Coding 2 | 30 /0.94 上0.04下0.10 |
3 | 10/15 Digestion INP His Coding 2 | 30 /0.94 上0.04下0.19 |
4 | 10/13 miniprep INP-His-CBDcex 6 | 30 /0.94 0.11 |
5 | 10/13 miniprep INP-His-CBDcex 6 | 30 /0.94 |
Uchino 16:08~
ABEF→centrifuge→resupend with PBS →centrifuge→through filter→resupend with PBS→through filter→centrifuge→respend with PBS→centrifuge→resupend with PBS→centrifuge→resupend with PBS
IJ Michimori
10/16
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder | |
2 | 10/15 colony PCR colony1 | |
3 | 10/15 colony PCR colony2 | |
4 | 10/15 colony PCR colony3 | |
5 | 10/15 colony PCR colony |
![](https://static.igem.org/mediawiki/2016/e/ef/T--Kyoto--10_16_No8_Electrophoresis_Sassa_10_17_12_16_22.1.jpg)
![](https://static.igem.org/mediawiki/2016/a/a2/T--Kyoto--10_16_No8_Electrophoresis_Sassa_10_17_12_16_22.2.jpg
)
10/16
PCR (Target)
SassaTemplates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
---|---|---|---|---|---|---|
2/22 scFv col.3 0.1 | VF 0.75 | VR 0.75 | KAPA HoFi 12.5 | 10.9 | 25 | |
2/22 scFv col.3 0.1 | VF 0.75 | VR 0.75 | KAPA HoFi 12.5 | 10.9 | 25 |
95℃ 5min
98℃ 20s×35
55℃ 15s ×35
72℃ 30s×35
4℃ infinity
10/16
Miniprep
Uchino, YamamotoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
10/15 Gel Extraction INP His coding 長 | 9.6 | 1.88 | 0.27 |
10/15 Gel Extraction INP His coding 短 | 74.4 | 1.87 | 0.84 |
10/15 Gel Extraction INP His CBDcex | 22.0 | 1.97 | 0.33 |
Insert MK | 64.5 | 1.92 | 1.17 |
Vector MK | 56.6 | 2.37 | 0.10 |
10/16
Ligation
YamamotoInsert | Vector | MilliQ | Ligation High |
---|---|---|---|
10/15 Insert MK/2µL | 10/15 Gel Extract INP His CBDcex 5.5µl | 0 | 7.5 |
10/15 Insert MK/1µL | 10/15 Gel Extract INP His CBDcex 6µL | 0 | 7.5 |
10/15 Insert MK/1µL | 10/15 Gel Extract INP His CBDcex 0.5µL | 6 | 7.5 |
10/15 Digestion INP His coding/5µL | 10/15 Digestion INP His CBDcex 2.5µL | 0 | 7.5 |
10/15 Digestion INP His coding/6µL | 10/15 Digestion INP His CBDcex 1.5µL | 0 | 7.5 |
10/15 Digestion INP His coding/4.5µL | 10/15 Digestion INP His CBDcex 3µL | 0 | 7.5 |
10/14Gel Extract INP His coding/5.5 | 10/15 Digestion INP His CBDcex 2µL | 0 | 7.5 |
10/14Gel Extract INP His coding/6 | 10/15 Digestion INP His CBDcex 1.5µL | 0 | 7.5 |
10/15 Digestion INP His coding 3.5µL | 10/15 Digestion INP His CBDcex 4µL | 0 | 7.5 |
10/15 Digestion INP His coding 2.5µL | 10/15 Digestion INP His CBDcex 5µL | 0 | 7.5 |
10/15 Digestion INP His coding 3.75 | 10/15 Digestion INP His CBDcex 3.75 | 0 | 7.5 |
10/16
Transformation
YamamotoSample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
---|---|---|---|
10/15Ligation Insert MK+Gel Extract1 2µL | 2 | DH5α/2 | |
10/15Ligation Insert MK+Gel Extract2 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion3 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion4 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion5 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion6 2µL | 2 | DH5α/2 | |
10/15Ligation Gel Extract+Digestion7 2µL | 2 | DH5α/2 | |
10/15Ligation Gel Extract+Digestion8 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion9 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion10 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion11 2µL | 2 | DH5α/2 | |
10/14 Ligation PuroductA 2µL | 2 | DH5α/2 | |
10/14 Ligation PuroductB 2µL | 2 | DH5α/2 |
10/16
PCR (Purification)
YamamotoSample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
---|---|---|---|
10/15 PCR scFv INP His codingFw+INP His RV | 284.4 | 1.88 | 2.18 |
10/16
Liquid Culture
YamamotoName | medium |
---|---|
10/14 Tranceformation INPHis coding2 colony1 | LB(CP) |
10/14 Tranceformation INPHis coding2 colony2 | LB(CP) |
10/16
Liquid Culture
YamamotoName | medium |
---|---|
10/14 Transformation INP His coding 2 colony1 | LB(CP) |
10/14 Transformation INP His coding 2 colony2 | LB(CP) |
10/16
fluorescence microscope assay
Uchinosample | number | magnification |
---|---|---|
cex bemliese | 13431 | ×200 |
cex bemliese | 13432~13441 | ×600 |
CTL bemliese | 13442 | ×200 |
CTL bemliese | 13443~13355 | ×600 |
10/16
Colony PCR
Matsumoto, Uchinonumber | sample | Any others | Any others(µL) |
---|---|---|---|
1~5 | 10/15Ligation Insert MK+Gel Extract1 | 2×Ready Mix | 12.5 |
6~10 | 10/15Ligation Insert MK+Gel Extract2 | Fwd primer | 1.25 |
11~15 | 10/15Ligation Insert MK+Gel Extract3 | Rev primer | 1.25 |
16~20 | 10/15Ligation Digestion+Digestion4 | MilliQ | 10 |
21~24 | 10/15Ligation Digestion+Digestion5 | 52×mix | |
25~28 | 10/15Ligation Digestion+Digestion6 | 2×Ready | 650 |
29~32 | 10/15Ligation Gel Extract+Digestion7 | Fwd | 65 |
33~36 | 10/15Ligation Gel Extract+Digestion8 | Rev | 65 |
37~40 | 10/15Ligation Digestion+Digestion9 | MilliQ | 500 |
41~44 | 10/15Ligation Digestion+Digestion10 | ||
45~48 | 10/15Ligation Digestion+Digestion11 |
×16
16Colony Picking DMSO 0.75 12.75 12
Mix 12.5 212.5 200
Fwd 1.25 21.25 18.75 20
Rev 1.25 21.25 18.75 20
MilliQ 9.25 157.25 138.75 148
16sample colony1~14・・・P15 sample colony
Blue 15・・・2/8 INP-His9
16・・・8/1 miniprep INPcex colony
10/16
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 1kb ladder | |
2 | 1 | |
3 | 2 | |
4 | 3 | |
5 | 4 | |
6 | 5 | |
7 | 6 | |
8 | 7 | |
9 | 8 | |
10 | 9 | |
11 | 10 | |
12 |
![](https://static.igem.org/mediawiki/2016/4/4d/T--Kyoto--10_16_No21_Electrophoresis_Sassa_10_17_10_54_10.jpg)
10/16
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | ||
2 | 1kb ladder | |
3 | 11 | |
4 | 12 | |
5 | 13 | |
6 | 14 | |
7 | 15 | |
8 | 16 | |
9 | 17 | |
10 | 18 | |
11 | 19 | |
12 | 20 |
![](https://static.igem.org/mediawiki/2016/7/76/T--Kyoto--10_16_No21_Electrophoresis_Sassa_10_17_10_54_18.jpg)
10/16
Fluorescence Microscopy
UchinoE.coli | OD600 | cellulose | size / weight | filter |
---|---|---|---|---|
- | - | bemliese | 5mm×5mm | - |
INPNC-His-ctl | 1 | bemliese | 5mm×5mm | - |
INPNC-His-CBDcex | 1 | bemliese | 5mm×5mm | - |
- | - | powder | 1 | filtered |
INPNC-His-ctl | 1 | powder | 1 | filtered |
INPNC-His-CBDcex | 1 | powder | 1 | filtered |
- | - | powder | 0.5 | filtered |
INPNC-His-ctl | 1 | powder | 0.5 | filtered |
INPNC-His-CBDcex | 1 | powder | 0.5 | filtered |
- | - | powder | 0.1 | filtered |
INPNC-His-ctl | 1 | powder | 0.1 | filtered |
INPNC-His-CBDcex | 1 | powder | 0.1 | filtered |
10/16
fluorescence microscope assay
Uchinosample | number | magnification |
---|---|---|
cex 1mg | 13456 | ×200 |
cex 1mg | 13457~13466 | ×600 |
cex 0.5mg | 13467 | ×200 |
cex 0.5mg | 13468~13378 | ×600 |
cex 0.5mg | 13379 | ×1000 |
CTL 1mg | 13380 | ×200 |
CTL 1mg | 13381~13390 | ×600 |
CTL 0.5mg | 13391 | ×200 |
CTL 0.5mg | 13392~13401 | ×600 |
CTL 0.5mg | 13402 | ×1000 |
cex 0.1mg | 13403 | ×200 |
cex 0.1mg | 13404,13407~13416 | ×600 |
cex 0.1mg | 13405,13406 | ×1000 |
CTL 0.1mg | 13417 | ×200 |
CTL 0.1mg | 13418~13430 | ×600 |
CTL 0.1mg | 13431 | ×200 |
cellulose 0.5mg | 13432~13441 | ×600 |
10/17
Electrophoresis
UchinoLane | sample/(µl) | Length(bp) |
---|---|---|
1 | 21 | |
2 | 22 | |
3 | 1kb ladder | |
4 | 23 | |
5 | 24 | |
6 | 25 | |
7 | 26 | |
8 | 27 | |
9 | 28 | |
10 | 29 | |
11 | 30 | |
12 |
![](https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--10_17_No22_Electrophoresis_Sassa_10_17_07_25_05.1.jpg)
![](https://static.igem.org/mediawiki/2016/9/96/T--Kyoto--10_17_No22_Electrophoresis_Sassa_10_17_07_25_05.2.jpg)
total 16mL
10/17
Miniprep
UchinoDNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
1 | 65.8 | 1.91 | 2.02 |
2 | 63.2 | 1.90 | 1.84 |
3 | 84.6 | 1.87 | 1.33 |
4 | 100.8 | 1.92 | 1.67 |
5 | 84.6 | 1.89 | 1.59 |
11 | 89.3 | 1.84 | 1.43 |
12 | 87.3 | 1.92 | 1.88 |
13 | 88.0 | 1.89 | 1.59 |
14 | 79.8 | 1.91 | 1.72 |
15 | 83.3 | 1.90 | 1.82 |
18 | 102.5 | 1.88 | 1.50 |
19 | 114.9 | 1.91 | 1.59 |
22 | 109.9 | 1.78 | 1.14 |
24 | 80.4 | 1.93 | 2.00 |
26 | 64.6 | 1.93 | 1.84 |
30 | 88.8 | 1.80 | 1.07 |
total 16mL
10/17
Colony PCR (typeA)
YamadaTemplates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
---|---|---|---|---|
10/15gel plated colony 1 | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
INPNC-His-scFv | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
INP-His-CBDcex | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
VF 1.25×8=10µL
VR 1.25×8=10µL
Kapa 12.5×8=100µL
DM80 0.75×8=6µL
MilliQ 9.25×8=74µL
Total 200µL