Dairyu6115 (Talk | contribs) |
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{{HokkaidoU_Japan}} | {{HokkaidoU_Japan}} | ||
{{Team:HokkaidoU_Japan/CSS}} | {{Team:HokkaidoU_Japan/CSS}} | ||
+ | {{HokkaidoU_Japan/footer}} | ||
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<body> | <body> | ||
<br> | <br> | ||
− | <img src="https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png" alt=" | + | <img src="https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png" alt="Protcols"> |
+ | |||
+ | <div align="right"><a href="https://2016.igem.org/Team:HokkaidoU_Japan/Notebook"><span class="big">Notebook</span></a></div> | ||
+ | |||
+ | |||
− | |||
<br> | <br> | ||
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<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
− | <td>Primer-F 10µM</td> | + | <td>Primer-F 10 µM</td> |
− | <td>Primer-R 10µM</td> | + | <td>Primer-R 10 µM</td> |
<td>MgSO<sub>4</sub></td> | <td>MgSO<sub>4</sub></td> | ||
<td>dNTPs</td> | <td>dNTPs</td> | ||
Line 47: | Line 51: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1</td> | <td>1</td> | ||
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<h2>2STEP Cycle (Tm value > 63°C)</h2> | <h2>2STEP Cycle (Tm value > 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
− | <td>3</td><td>68</td><td> | + | <td>3</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~3 × (25~45) |
<h2>3STEP Cycle (Tm value < 63°C)</h2> | <h2>3STEP Cycle (Tm value < 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>Tm</td><td>30</td> | <td>3</td><td>Tm</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
− | <td>4</td><td>68</td><td> | + | <td>4</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>5</td><td>4</td><td>Hold</td> | <td>5</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~4 × (25~45) |
<h2>STEP DOWN</h2> | <h2>STEP DOWN</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
− | <td>3</td><td>74</td><td> | + | <td>3</td><td>74</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>98</td><td>10</td> | <td>4</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>5</td><td>72</td><td>30</td> | <td>5</td><td>72</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>6</td><td>98</td><td>10</td> | <td>6</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>7</td><td>70</td><td>30</td> | <td>7</td><td>70</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>8</td><td>98</td><td>10</td> | <td>8</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>9</td><td>68</td><td>30</td> | <td>9</td><td>68</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>10</td><td>68</td><td>420</td> | <td>10</td><td>68</td><td>420</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>11</td><td>4</td><td>Hold</td> | <td>11</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
Cycle: | Cycle: | ||
− | <br> | + | <br>sequence 2~3 × 5 |
− | <br> | + | <br>sequence 4~5 × 5 |
− | <br> | + | <br>sequence 6~7 × 5 |
− | <br> | + | <br>sequence 8~9 × 15 |
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp1").style.display="none"; | document.getElementById("disp1").style.display="none"; | ||
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</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp2").style.display="none"; | document.getElementById("disp2").style.display="none"; | ||
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Mix the following reagents in PCR tube. | Mix the following reagents in PCR tube. | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
− | <td>RE1 | + | <td>RE1 10 U/µL</td> |
− | <td>RE2 | + | <td>RE2 10 U/µL</td> |
<td>Appropriate buffer</td> | <td>Appropriate buffer</td> | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>16</td> | <td>16</td> | ||
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</table> | </table> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>37</td><td>120</td> | <td>1</td><td>37</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>65</td><td>15</td> | <td>2</td><td>65</td><td>15</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>4</td><td>Hold</td> | <td>3</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
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<div id="disp4"> | <div id="disp4"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
− | Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit | + | Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer. |
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td> | <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td> | <th>Volume (µL)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td> | ||
</tr> | </tr> | ||
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Thermal protocol is following | Thermal protocol is following | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>16</td><td>30</td> | <td>1</td><td>16</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>65</td><td>10</td> | <td>2</td><td>65</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>4</td><td>Hold</td> | <td>3</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp4").style.display="none"; | document.getElementById("disp4").style.display="none"; | ||
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<ol> | <ol> | ||
<li>Put gel into electrophoresis tank.</li> | <li>Put gel into electrophoresis tank.</li> | ||
− | <li> | + | <li>Pour 2x TBE buffer into the tank to soak gel.</li> |
− | <li>Add 5 µL of EtBr into | + | <li>Add 5 µL of EtBr into cathode.</li> |
<li>Pre-migration for 30 min at 100 V.</li> | <li>Pre-migration for 30 min at 100 V.</li> | ||
− | <li>Apply DNA | + | <li>Apply DNA solutions with 6x loading dye and ladder.</li> |
<li>Start electrophoresis at 100 V.</li> | <li>Start electrophoresis at 100 V.</li> | ||
+ | <li>Stop at appropriate time. | ||
</ol> | </ol> | ||
</span> | </span> | ||
− | |||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp5").style.display="none"; | document.getElementById("disp5").style.display="none"; | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp6").style.display="none"; | document.getElementById("disp6").style.display="none"; | ||
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<li>Remove supernatant and add 600 µL of 70% ethanol.</li> | <li>Remove supernatant and add 600 µL of 70% ethanol.</li> | ||
<li>Centrifuge at 15,000 rpm for 5 min at 25°C.</li> | <li>Centrifuge at 15,000 rpm for 5 min at 25°C.</li> | ||
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up at room temperature with light sheilding.</li> |
<li>Suspend with 10 µL of TE.</li> | <li>Suspend with 10 µL of TE.</li> | ||
</ol> | </ol> | ||
− | |||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp7").style.display="none"; | document.getElementById("disp7").style.display="none"; | ||
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<span class="nomal2"> | <span class="nomal2"> | ||
<table> | <table> | ||
− | <tr> | + | |
− | + | <tr align="center"> | |
+ | |||
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
− | <td | + | <td>Kapa-Taq (Taq polymerase)</td> |
− | <td | + | <td>EX-F primer 10 µM</td> |
− | <td | + | <td>PS-R primer 10 µM</td> |
<td>Total</td> | <td>Total</td> | ||
− | |||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
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<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>94</td><td>30</td> | <td>2</td><td>94</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
− | <td>3</td><td>68</td><td>60 | + | <td>3</td><td>68</td><td>60 / 1kbp</td> |
</tr> | </tr> | ||
+ | <tr align="center"> | ||
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycles: | + | Cycles: sequence 2~3 × 25~45 |
− | + | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp8").style.display="none"; | document.getElementById("disp8").style.display="none"; | ||
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<span class="nomal2"> | <span class="nomal2"> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>5 x Sequencing Buffer</td> | <td>5 x Sequencing Buffer</td> | ||
− | <td>primer 1µM</td> | + | <td>primer 1 µM</td> |
<td>template DNA</td> | <td>template DNA</td> | ||
<td>Ready Reaction Premix</td> | <td>Ready Reaction Premix</td> | ||
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<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1.5</td> | <td>1.5</td> | ||
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<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>96</td><td>10</td> | <td>1</td><td>96</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>50</td><td>5</td> | <td>2</td><td>50</td><td>5</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>60</td><td>240</td> | <td>3</td><td>60</td><td>240</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~4 × 25 |
<h2>Ethanol precipitation</h2> | <h2>Ethanol precipitation</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>PCR product</td> | <td>PCR product</td> | ||
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<td>100% EtOH</td> | <td>100% EtOH</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>10</td> | <td>10</td> | ||
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</table> | </table> | ||
<ol> | <ol> | ||
− | <li>Centrifuge at 15,000 rpm for 15 min at room temprature</li> | + | <li>Centrifuge at 15,000 rpm for 15 min at room temprature.</li> |
− | <li>Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.</li> | + | <li>Remove supernatant, add 100 µL of 70% EtOH and tap tubes by finger.</li> |
− | <li>Centrifuge at 15,000 rpm for 10 min at room temprature</li> | + | <li>Centrifuge at 15,000 rpm for 10 min at room temprature.</li> |
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up at room temperature.</li> |
<li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li> | <li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li> | ||
<li>Set the plate and start electrophoresis.</li> | <li>Set the plate and start electrophoresis.</li> | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp9").style.display="none"; | document.getElementById("disp9").style.display="none"; | ||
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<li>Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.</li> | <li>Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.</li> | ||
<li>Incubate the cells at 130 rpm at 20°C, until OD<sub>600</sub> reach 0.5.</li> | <li>Incubate the cells at 130 rpm at 20°C, until OD<sub>600</sub> reach 0.5.</li> | ||
− | <li>Take 50 mL of incubated cells to two | + | <li>Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.</li> |
<li>Remove supernatant and add 75 mL of TB to each tube.</li> | <li>Remove supernatant and add 75 mL of TB to each tube.</li> | ||
<li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.</li> | <li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.</li> | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp10").style.display="none"; | document.getElementById("disp10").style.display="none"; | ||
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<li>Incubate on ice for 30 min.</li> | <li>Incubate on ice for 30 min.</li> | ||
<li>Add to LB.</li> | <li>Add to LB.</li> | ||
− | <li> | + | <li>Incubate the cells for 2 hrs at 37°C.</li> |
<li>Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.</li> | <li>Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.</li> | ||
<li>Incubate the plate(s) at 37°C for 16~20 hrs.</li> | <li>Incubate the plate(s) at 37°C for 16~20 hrs.</li> | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp11").style.display="none"; | document.getElementById("disp11").style.display="none"; | ||
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</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp12").style.display="none"; | document.getElementById("disp12").style.display="none"; | ||
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<li>Pick the colony with an inoculating loop from the agar plate.</li> | <li>Pick the colony with an inoculating loop from the agar plate.</li> | ||
<li>Drag the loop across on a new agar plate.</li> | <li>Drag the loop across on a new agar plate.</li> | ||
− | <li> | + | <li>Resterilize the loop and drag it across again.</li> |
<li>Repeat method 3.</li> | <li>Repeat method 3.</li> | ||
</ol> | </ol> | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp13").style.display="none"; | document.getElementById("disp13").style.display="none"; | ||
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<li>Remove supernatant and add 100 µL of 70% ethanol.</li> | <li>Remove supernatant and add 100 µL of 70% ethanol.</li> | ||
<li>Centrifuge at 15,000 rpm for 2 min at 4°C.</li> | <li>Centrifuge at 15,000 rpm for 2 min at 4°C.</li> | ||
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up at room temperature with light sheilding.</li> |
<li>Suspend with 10 µL of TE.</li> | <li>Suspend with 10 µL of TE.</li> | ||
</ol> | </ol> | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp14").style.display="none"; | document.getElementById("disp14").style.display="none"; | ||
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<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
− | <td>5'-biotinylated 100-UP primer 10µM</td> | + | <td>5'-biotinylated 100-UP primer 10 µM</td> |
− | <td>5'-biotinylated 200-DN primer 10µM</td> | + | <td>5'-biotinylated 200-DN primer 10 µM</td> |
<td>MgSO<sub>4</sub></td> | <td>MgSO<sub>4</sub></td> | ||
<td>dNTPs</td> | <td>dNTPs</td> | ||
Line 725: | Line 741: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1</td> | <td>1</td> | ||
Line 741: | Line 757: | ||
<h2>2STEP Cycle (Tm value > 63°C)</h2> | <h2>2STEP Cycle (Tm value > 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
− | <td>3</td><td>68</td><td> | + | <td>3</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~3 × (25~45) |
<h2>Preparation of magnetic beads</h2> | <h2>Preparation of magnetic beads</h2> | ||
Line 785: | Line 801: | ||
</span> | </span> | ||
</div> | </div> | ||
− | < | + | |
+ | <br> | ||
+ | <br> | ||
<script> | <script> | ||
document.getElementById("disp15").style.display="none"; | document.getElementById("disp15").style.display="none"; | ||
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</script> | </script> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <!-- 2016contents 閉じる --> | ||
+ | |||
+ | <!--begin footer--> | ||
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+ | <a href="https://twitter.com/igem_hokkaidou"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/1/13/T--HokkaidoU_Japan--twitter.png" alt="Twitter"></a> | ||
+ | |||
+ | <a href="mailto:igemhokkaidou@gmail.com"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/4/4e/T--HokkaidoU_Japan--mail.png" alt="E-mail"></a> | ||
+ | |||
+ | <a href="http://igemhokkaidou.wordpress.com"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/3/3b/T--HokkaidoU_Japan--icon1.png" alt="Web"> | ||
+ | </a> | ||
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</body> | </body> | ||
</html> | </html> |
Latest revision as of 19:25, 19 October 2016
Team:HokkaidoU Japan
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Cycles: sequence 2~3 × 25~45
Cycle: sequence 2~4 × 25
Thermal protocol is following
Cycle: sequence 2~3 × (25~45)
![Protcols](https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png)
PCR
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Thermal protocol is following
Cycle: sequence 2~3 × (25~45)
Cycle: sequence 2~4 × (25~45)
Cycle:
sequence 2~3 × 5
sequence 4~5 × 5
sequence 6~7 × 5
sequence 8~9 × 15
Solution | template DNA | Primer-F 10 µM | Primer-R 10 µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1 | 1 | 3 | 5 | 5 | 1 | 33 | 50 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30 / 1kbp |
4 | 4 | Hold |
3STEP Cycle (Tm value < 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | Tm | 30 |
4 | 68 | 30 / 1kbp |
5 | 4 | Hold |
STEP DOWN
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 74 | 30 / 1kbp |
4 | 98 | 10 |
5 | 72 | 30 |
6 | 98 | 10 |
7 | 70 | 30 |
8 | 98 | 10 |
9 | 68 | 30 |
10 | 68 | 420 |
11 | 4 | Hold |
sequence 2~3 × 5
sequence 4~5 × 5
sequence 6~7 × 5
sequence 8~9 × 15
PCR Purification
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products
Purification of PCR products
Digestion
Mix the following reagents in PCR tube.
Solution | DNA | RE1 10 U/µL | RE2 10 U/µL | Appropriate buffer | Total |
---|---|---|---|---|---|
Volume (µL) | 16 | 1 | 1 | 2 | 20 |
Sequence | Temp. (°C) | Time (min) |
---|---|---|
1 | 37 | 120 |
2 | 65 | 15 |
3 | 4 | Hold |
Ligation
Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer.
Thermal protocol is following
Solution | Vector DNA | Insert DNA | DW | Mighty Mix | Total |
---|---|---|---|---|---|
Volume (µL) | 1 | 2 | 2 | 5 | 10 |
Sequence | Temp. (°C) | Time (min) |
---|---|---|
1 | 16 | 30 |
2 | 65 | 10 |
3 | 4 | Hold |
Electrophoresis
- Put gel into electrophoresis tank.
- Pour 2x TBE buffer into the tank to soak gel.
- Add 5 µL of EtBr into cathode.
- Pre-migration for 30 min at 100 V.
- Apply DNA solutions with 6x loading dye and ladder.
- Start electrophoresis at 100 V.
- Stop at appropriate time.
Gel Extraction
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
DNA extraction from gel
Ethanol precipitation
- Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
- Leave it at room temperature for 5 min.
- Centrifuge at 15,000 rpm for 15 min at 25°C.
- Remove supernatant and add 600 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 5 min at 25°C.
- Remove supernatant and dry up at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Colony PCR
Solution | DNA | Kapa-Taq (Taq polymerase) | EX-F primer 10 µM | PS-R primer 10 µM | Total |
---|---|---|---|---|---|
Volume (µL) | 4.2 | 5 | 0.4 | 0.4 | 10 |
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 60 / 1kbp |
4 | 4 | Hold |
Sequencing
Solution | 5 x Sequencing Buffer | primer 1 µM | template DNA | Ready Reaction Premix | DW | Total |
---|---|---|---|---|---|---|
Volume (µL) | 1.5 | 1.5 | 1 | 1 | 5 | 10 |
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 96 | 10 |
2 | 50 | 5 |
3 | 60 | 240 |
4 | 4 | Hold |
Ethanol precipitation
Solution | PCR product | DW | 3M NaOAc | Glycogen | 100% EtOH |
---|---|---|---|---|---|
Volume (µL) | 10 | 10 | 2 | 1 | 50 |
- Centrifuge at 15,000 rpm for 15 min at room temprature.
- Remove supernatant, add 100 µL of 70% EtOH and tap tubes by finger.
- Centrifuge at 15,000 rpm for 10 min at room temprature.
- Remove supernatant and dry up at room temperature.
- Resuspend the pellet to HiDi formamide and remove to 96-well plate.
- Set the plate and start electrophoresis.
Competent Cells
- Thaw original competent cells on ice.
- Add 5 µL of original competent cells to 2 mL of LB.
- Incubate the cells for 16 hrs at 37°C.
- Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
- Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
- Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 75 mL of TB to each tube.
- Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 32 mL of TB.
- Add 32 µL of DMSO 10 times.
- Take 50 µL and freeze with liquid nitrogen.
Transformation
- Add plasmid to thawed competent cells on ice.
- Incubate on ice for 30 min.
- Add to LB.
- Incubate the cells for 2 hrs at 37°C.
- Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
- Incubate the plate(s) at 37°C for 16~20 hrs.
Mini-prep
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
fast / standard / low copy protocol
fast / standard / low copy protocol
Streaking (Single colony isolation)
- Pick the colony with an inoculating loop from the agar plate.
- Drag the loop across on a new agar plate.
- Resterilize the loop and drag it across again.
- Repeat method 3.
PEG precipitation
- Add 13 µL of PEG to 20 µL of product(s).
- Leave it at room temperature for 1 hr.
- Centrifuge at 15,000 rpm for 20 min at 4°C.
- Remove supernatant and add 100 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 2 min at 4°C.
- Remove supernatant and dry up at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Gel Free System
Preparation of biotinylated DNA fragments
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.Solution | template DNA | 5'-biotinylated 100-UP primer 10 µM | 5'-biotinylated 200-DN primer 10 µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1.5 | 1.5 | 3 | 5 | 5 | 1 | 32 | 50 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30 / 1kbp |
4 | 4 | Hold |
Preparation of magnetic beads
- Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
- Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
- Remove supernatant.
Fixation to magnetic beads
- Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
- Mix by vibration using sonic-toothbrush.
- Collect the beads using magnet.
- Remove supernatant containing excess amount of free DNA fragment.
Double restriction digestion
- Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
- Mix by pumping using pipette.
- Incubate at 37 °C for 30 min.
- Collect the beads using magnet.
- Obtain supernatant containing digested DNA fragment.
- Purify the supernatant by ethanol precipitation.