Difference between revisions of "Team:HokkaidoU Japan/Experiments"

 
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{{HokkaidoU_Japan}}
 
{{HokkaidoU_Japan}}
 
{{Team:HokkaidoU_Japan/CSS}}
 
{{Team:HokkaidoU_Japan/CSS}}
 +
{{HokkaidoU_Japan/footer}}
  
  
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left: 30px;
 
left: 30px;
 
}
 
}
 +
  
 
</style>
 
</style>
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<body>
 
<body>
 
<br>
 
<br>
−
<img src="https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png" alt="Prptpcols">
+
<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>
 +
 
 +
 
  
−
<div class="ichi">
 
  
 
<br>
 
<br>
−
<h2 onClick="hyoji1()"><span class="ka-soru">PCR</span></h2>
+
<h1 onClick="hyoji1()"><span class="ka-soru">PCR</span></h1>
  
  
 
<div id="disp1">
 
<div id="disp1">
−
 
+
<span class="nomal2">
 
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
 
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
  
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Solution</th>
 
     <th>Solution</th>
 
     <td>template DNA</td>
 
     <td>template DNA</td>
−
     <td>Primer-F 10&micro;M</td>
+
     <td>Primer-F 10 &micro;M</td>
−
     <td>Primer-R 10&micro;M</td>
+
     <td>Primer-R 10 &micro;M</td>
 
     <td>MgSO<sub>4</sub></td>
 
     <td>MgSO<sub>4</sub></td>
 
     <td>dNTPs</td>
 
     <td>dNTPs</td>
Line 46: Line 51:
 
     <td>Total</td>
 
     <td>Total</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>1</td>
 
     <td>1</td>
Line 59: Line 64:
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<p>Thermal protocol is following</p>
+
Thermal protocol is following
−
<h3>2STEP Cycle (Tm value &gt; 63&deg;C)</h3>
+
<h2>2STEP Cycle (Tm value &gt; 63&deg;C)</h2>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>30sec / 1kbp</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>
−
<p>Cycle: sequence2~3 &times; (25~45)</p>
+
Cycle: sequence 2~3 &times; (25~45)
  
−
<h3>3STEP Cycle (Tm value &lt; 63&deg;C)</h3>
+
<h2>3STEP Cycle (Tm value &lt; 63&deg;C)</h2>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>30sec / 1kbp</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>
−
<p>Cycle: sequence2~4 &times; (25~45)</p>
+
Cycle: sequence 2~4 &times; (25~45)
  
−
<h3>STEP DOWN</h3>
+
<h2>STEP DOWN</h2>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>30sec / 1kbp</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>
  
 
</table>
 
</table>
−
<p>Cycle:sequence2~3 &times; (5)</p>
+
Cycle:
−
<p>   sequence4~5 &times; (5)</p>
+
<br>sequence 2~3 &times; 5
−
<p>   sequence6~7 &times; (5)</p>
+
<br>sequence 4~5 &times; 5
−
<p>   sequence8~9 &times; (15)</p>
+
<br>sequence 6~7 &times; 5
−
 
+
<br>sequence 8~9 &times; 15
  
 +
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp1").style.display="none";
 
document.getElementById("disp1").style.display="none";
Line 171: Line 178:
  
  
−
<h2 onClick="hyoji2()"><span class="ka-soru">PCR Purification</span></h2>
+
<h1 onClick="hyoji2()"><span class="ka-soru">PCR Purification</span></h1>
  
 
<div id="disp2">
 
<div id="disp2">
 +
<span class="nomal2">
 +
FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
 +
<br>Purification of PCR products
 +
</span>
  
−
<p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
+
</div>
−
<br>Purification of PCR products</p>
+
  
−
 
−
</div>
 
 
<script>
 
<script>
 
document.getElementById("disp2").style.display="none";
 
document.getElementById("disp2").style.display="none";
Line 198: Line 206:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji3()"><span class="ka-soru">Digestion</span></h2>
+
<h1 onClick="hyoji3()"><span class="ka-soru">Digestion</span></h1>
 
<div id="disp3">
 
<div id="disp3">
−
 
+
<span class="nomal2">
−
<p>Mix the following reagents in PCR tube.</p>
+
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 10U/&micro;L</td>
+
     <td>RE1 10 U/&micro;L</td>
−
     <td>RE2 10U/&micro;L</td>
+
     <td>RE2 10 U/&micro;L</td>
 
     <td>Appropriate buffer</td>
 
     <td>Appropriate buffer</td>
 
     <td>Total</td>
 
     <td>Total</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>16</td>
 
     <td>16</td>
Line 221: Line 229:
 
</table>
 
</table>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>
 
</table>
 
</table>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp3").style.display="none";
 
document.getElementById("disp3").style.display="none";
Line 254: Line 263:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji4()"><span class="ka-soru">Ligation</span></h2>
+
 
 +
<h1 onClick="hyoji4()"><span class="ka-soru">Ligation</span></h1>
 
<div id="disp4">
 
<div id="disp4">
−
<p>
+
<span class="nomal2">
−
  Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit &lt;Mighty Mix&amp;rt; (Takara Bio Inc.) which contains ligase and buffer.
+
Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer.
−
</p>
+
 
 
<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 (&micro;L)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td>
 
     <th>Volume (&micro;L)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<p>Thermal protocol is following</p>
+
Thermal protocol is following
−
<table
+
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>
 
</table>
 
</table>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp4").style.display="none";
 
document.getElementById("disp4").style.display="none";
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</script>
 
</script>
  
−
<h2 onClick="hyoji5()"><span class="ka-soru">Electrophoresis</span></h2>
+
<h1 onClick="hyoji5()"><span class="ka-soru">Electrophoresis</span></h1>
 
<div id="disp5">
 
<div id="disp5">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
   <li>Put gel into electrophoresis tank.</li>
 
   <li>Put gel into electrophoresis tank.</li>
−
   <li>Pore 2x TBE buffer into the tank to soak gel.</li>
+
   <li>Pour 2x TBE buffer into the tank to soak gel.</li>
−
   <li>Add 5  &micro;L  of EtBr into cathod.</li>
+
   <li>Add 5  &micro;L  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 solution with 6x loading dye and ladder.</li>
+
   <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>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp5").style.display="none";
 
document.getElementById("disp5").style.display="none";
Line 333: Line 347:
  
  
−
<h2 onClick="hyoji6()"><span class="ka-soru">Gel Extraction</span></h2>
+
<h1 onClick="hyoji6()"><span class="ka-soru">Gel Extraction</span></h1>
 
<div id="disp6">
 
<div id="disp6">
 +
<span class="nomal2">
 +
FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
 +
<br>DNA extraction from gel
  
−
<p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
+
</span>
−
<br>DNA extraction from gel</p>
+
−
 
+
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp6").style.display="none";
 
document.getElementById("disp6").style.display="none";
Line 359: Line 375:
  
  
−
<h2 onClick="hyoji7()"><span class="ka-soru">Ethanol precipitation</span></h2>
+
<h1 onClick="hyoji7()"><span class="ka-soru">Ethanol precipitation</span></h1>
 
<div id="disp7">
 
<div id="disp7">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
   <li>Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.</li>
 
   <li>Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.</li>
Line 367: Line 384:
 
   <li>Remove supernatant and add  600 &micro;L  of 70% ethanol.</li>
 
   <li>Remove supernatant and add  600 &micro;L  of 70% ethanol.</li>
 
   <li>Centrifuge at 15,000 rpm for 5 min at 25&deg;C.</li>
 
   <li>Centrifuge at 15,000 rpm for 5 min at 25&deg;C.</li>
−
   <li>Remove supernatant and air-dry at room temperature with light sheilding.</li>
+
   <li>Remove supernatant and dry up at room temperature with light sheilding.</li>
 
   <li>Suspend with 10  &micro;L  of TE.</li>
 
   <li>Suspend with 10  &micro;L  of TE.</li>
 
</ol>
 
</ol>
 +
</span>
 +
</div>
  
−
 
−
</div>
 
 
<script>
 
<script>
 
document.getElementById("disp7").style.display="none";
 
document.getElementById("disp7").style.display="none";
Line 391: Line 408:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji8()"><span class="ka-soru">Colony PCR</span></h2>
+
<h1 onClick="hyoji8()"><span class="ka-soru">Colony PCR</span></h1>
 
<div id="disp8">
 
<div id="disp8">
 +
<span class="nomal2">
 
<table>
 
<table>
−
   <tr>
+
 
 +
   <tr align="center">
 +
 
 
     <th>Solution</th>
 
     <th>Solution</th>
 
     <td>DNA</td>
 
     <td>DNA</td>
 
     <td>Kapa-Taq (Taq polymerase)</td>
 
     <td>Kapa-Taq (Taq polymerase)</td>
−
     <td>EX-F primer 10&micro;M</td>
+
     <td>EX-F primer 10 &micro;M</td>
−
     <td>PS-R primer 10&micro;M</td>
+
     <td>PS-R primer 10 &micro;M</td>
 
     <td>Total</td>
 
     <td>Total</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>4.2</td>
 
     <td>4.2</td>
Line 413: Line 433:
  
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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 sec / 1kbp</td>
+
     <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>
−
<p>Cycles: sequence2~3 &times; 25~45</p>
+
Cycles: sequence 2~3 &times; 25~45
 +
</span>
 +
</div>
  
−
 
−
</div>
 
 
<script>
 
<script>
 
document.getElementById("disp8").style.display="none";
 
document.getElementById("disp8").style.display="none";
Line 450: Line 471:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji9()"><span class="ka-soru">Sequencing</span></h2>
+
<h1 onClick="hyoji9()"><span class="ka-soru">Sequencing</span></h1>
 
<div id="disp9">
 
<div id="disp9">
 +
<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 1&micro;M</td>
+
     <td>primer 1 &micro;M</td>
 
     <td>template DNA</td>
 
     <td>template DNA</td>
 
     <td>Ready Reaction Premix</td>
 
     <td>Ready Reaction Premix</td>
Line 462: Line 484:
 
     <td>Total</td>
 
     <td>Total</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>1.5</td>
 
     <td>1.5</td>
Line 474: Line 496:
  
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>
−
<p>Cycle: sequence2~4 &times; 25</p>
+
Cycle: sequence 2~4 &times; 25
  
  
−
<h3>Ethanol precipitation</h3>
+
<h2>Ethanol precipitation</h2>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Solution</th>
 
     <th>Solution</th>
 
     <td>PCR product</td>
 
     <td>PCR product</td>
Line 503: Line 525:
 
     <td>100% EtOH</td>
 
     <td>100% EtOH</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>10</td>
 
     <td>10</td>
Line 513: Line 535:
 
</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 &micro;L  of 70% EtOH and tap tubes by finger.</li>
+
   <li>Remove supernatant, add 100 &micro;L  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 air dry at room temperature.</li>
+
   <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>
 
</ol>
 
</ol>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp9").style.display="none";
 
document.getElementById("disp9").style.display="none";
Line 541: Line 564:
  
  
−
<h2 onClick="hyoji10()"><span class="ka-soru">Competent Cells</span></h2>
+
<h1 onClick="hyoji10()"><span class="ka-soru">Competent Cells</span></h1>
 
<div id="disp10">
 
<div id="disp10">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
<li>Thaw original competent cells on ice.</li>
 
<li>Thaw original competent cells on ice.</li>
Line 549: Line 573:
 
<li>Add 5 &micro;L, 50 &micro;L, and 500 &micro;L of original cells to 100 mL of LB.</li>
 
<li>Add 5 &micro;L, 50 &micro;L, and 500 &micro;L of original cells to 100 mL of LB.</li>
 
<li>Incubate the cells at 130 rpm at 20&deg;C, until OD<sub>600</sub> reach 0.5.</li>
 
<li>Incubate the cells at 130 rpm at 20&deg;C, until OD<sub>600</sub> reach 0.5.</li>
−
<li>Take 50 mL of incubated cells to two differnt culture tubes and centrifuge them at 3,000 rpm for 20 min at 4&deg;C.</li>
+
<li>Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4&deg;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&deg;C.</li>
 
<li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4&deg;C.</li>
Line 556: Line 580:
 
<li>Take 50 &micro;L and freeze with liquid nitrogen.</li>
 
<li>Take 50 &micro;L and freeze with liquid nitrogen.</li>
 
</ol>
 
</ol>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp10").style.display="none";
 
document.getElementById("disp10").style.display="none";
Line 576: Line 601:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji11()"><span class="ka-soru">Transformation</span></h2>
+
<h1 onClick="hyoji11()"><span class="ka-soru">Transformation</span></h1>
 
<div id="disp11">
 
<div id="disp11">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
   <li>Add plasmid to thawed competent cells on ice.</li>
 
   <li>Add plasmid to thawed competent cells on ice.</li>
 
   <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>(Incubate the cells for 2 hrs at 37&deg;C.)</li>
+
   <li>Incubate the cells for 2 hrs at 37&deg;C.</li>
 
   <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
 
   <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
 
   <li>Incubate the plate(s) at 37&deg;C for 16~20 hrs.</li>
 
   <li>Incubate the plate(s) at 37&deg;C for 16~20 hrs.</li>
 
</ol>
 
</ol>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp11").style.display="none";
 
document.getElementById("disp11").style.display="none";
Line 606: Line 633:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji12()"><span class="ka-soru">Mini-prep</span></h2>
+
<h1 onClick="hyoji12()"><span class="ka-soru">Mini-prep</span></h1>
 
<div id="disp12">
 
<div id="disp12">
−
<p>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd)
+
<span class="nomal2">
−
<br>fast / standard / low copy protocol</p>
+
FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd)
−
 
+
<br>fast / standard / low copy protocol
 +
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp12").style.display="none";
 
document.getElementById("disp12").style.display="none";
Line 630: Line 659:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji13()"><span class="ka-soru">Streaking (Single colony isolation)</span></h2>
+
<h1 onClick="hyoji13()"><span class="ka-soru">Streaking (Single colony isolation)</span></h1>
 
<div id="disp13">
 
<div id="disp13">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
   <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>Re-sterilise the loop and drag it across again.</li>
+
   <li>Resterilize the loop and drag it across again.</li>
 
   <li>Repeat method 3.</li>
 
   <li>Repeat method 3.</li>
 
</ol>
 
</ol>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp13").style.display="none";
 
document.getElementById("disp13").style.display="none";
Line 658: Line 689:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji14()"><span class="ka-soru">PEG precipitation</span></h2>
+
<h1 onClick="hyoji14()"><span class="ka-soru">PEG precipitation</span></h1>
 
<div id="disp14">
 
<div id="disp14">
 +
<span class="nomal2">
 
<ol>
 
<ol>
 
   <li>Add 13  &micro;L  of PEG to 20  &micro;L  of product(s).</li>
 
   <li>Add 13  &micro;L  of PEG to 20  &micro;L  of product(s).</li>
Line 666: Line 698:
 
   <li>Remove supernatant and add  100 &micro;L  of 70% ethanol.</li>
 
   <li>Remove supernatant and add  100 &micro;L  of 70% ethanol.</li>
 
   <li>Centrifuge at 15,000 rpm for 2 min at 4&deg;C.</li>
 
   <li>Centrifuge at 15,000 rpm for 2 min at 4&deg;C.</li>
−
   <li>Remove supernatant and air-dry at room temperature with light sheilding.</li>
+
   <li>Remove supernatant and dry up at room temperature with light sheilding.</li>
 
   <li>Suspend with 10  &micro;L  of TE.</li>
 
   <li>Suspend with 10  &micro;L  of TE.</li>
 
</ol>
 
</ol>
−
 
+
</span>
 
</div>
 
</div>
 +
 
<script>
 
<script>
 
document.getElementById("disp14").style.display="none";
 
document.getElementById("disp14").style.display="none";
Line 689: Line 722:
 
</script>
 
</script>
  
−
<h2 onClick="hyoji15()"><span class="ka-soru">Gel Free System</span></h2>
+
<h1 onClick="hyoji15()"><span class="ka-soru">Gel Free System</span></h1>
 
<div id="disp15">
 
<div id="disp15">
−
<h3>Preparation of biotinylated DNA fragments</h3>
+
<span class="nomal2">
−
<p>
+
<h2>Preparation of biotinylated DNA fragments</h2>
−
  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.
+
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.
−
</p>
+
 
 
<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 10&micro;M</td>
+
     <td>5'-biotinylated 100-UP primer 10 &micro;M</td>
−
     <td>5'-biotinylated 200-DN primer 10&micro;M</td>
+
     <td>5'-biotinylated 200-DN primer 10 &micro;M</td>
 
     <td>MgSO<sub>4</sub></td>
 
     <td>MgSO<sub>4</sub></td>
 
     <td>dNTPs</td>
 
     <td>dNTPs</td>
Line 708: Line 741:
 
     <td>Total</td>
 
     <td>Total</td>
 
   </tr>
 
   </tr>
−
   <tr>
+
   <tr align="center">
 
     <th>Volume (&micro;L)</th>
 
     <th>Volume (&micro;L)</th>
 
     <td>1</td>
 
     <td>1</td>
Line 721: Line 754:
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<p>Thermal protocol is following</p>
+
Thermal protocol is following
−
<h3>2STEP Cycle (Tm value &gt; 63&deg;C)</h3>
+
<h2>2STEP Cycle (Tm value &gt; 63&deg;C)</h2>
 
<table>
 
<table>
−
   <tr>
+
   <tr align="center">
 
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 
     <th>Sequence</th><th>Temp. (&deg;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>30sec / 1kbp</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>
−
<p>Cycle: sequence2~3 &times; (25~45)</p>
+
Cycle: sequence 2~3 &times; (25~45)
  
−
<h3>Preparation of magnetic beads</h3>
+
<h2>Preparation of magnetic beads</h2>
 
<ol>
 
<ol>
 
   <li>Mix 2 &micro;L  of magnetic beads (SiMAG-Streptavidin) and 48 &micro;L  of TE by vibration using sonic-toothbrush.</li>
 
   <li>Mix 2 &micro;L  of magnetic beads (SiMAG-Streptavidin) and 48 &micro;L  of TE by vibration using sonic-toothbrush.</li>
Line 749: Line 782:
 
</ol>
 
</ol>
  
−
<h3>Fixation to magnetic beads</h3>
+
<h2>Fixation to magnetic beads</h2>
 
<ol>
 
<ol>
 
   <li>Add 3 &micro;L  of PCR product (0.48 pmol) and 7 &micro;L  of TE to beads.</li>
 
   <li>Add 3 &micro;L  of PCR product (0.48 pmol) and 7 &micro;L  of TE to beads.</li>
Line 757: Line 790:
 
</ol>
 
</ol>
  
−
<h3>Double restriction digestion</h3>
+
<h2>Double restriction digestion</h2>
 
<ol>
 
<ol>
 
   <li>Add Digestion Premix containing 1 &micro;L  of 10x RE solution, 8 &micro;L  of DW and each 0.5 &micro;L  of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li>
 
   <li>Add Digestion Premix containing 1 &micro;L  of 10x RE solution, 8 &micro;L  of DW and each 0.5 &micro;L  of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li>
Line 766: Line 799:
 
   <li>Purify the supernatant by ethanol precipitation.</li>
 
   <li>Purify the supernatant by ethanol precipitation.</li>
 
</ol>
 
</ol>
−
 
+
</span>
−
</div>
+
 
</div>
 
</div>
 +
 +
<br>
 +
<br>
 
<script>
 
<script>
 
document.getElementById("disp15").style.display="none";
 
document.getElementById("disp15").style.display="none";
Line 787: Line 822:
 
</script>
 
</script>
  
 +
 +
 +
</div>
 +
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Latest revision as of 19:25, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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Protcols


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.
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
Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
36830 / 1kbp
44Hold
Cycle: sequence 2~3 × (25~45)

3STEP Cycle (Tm value < 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
3Tm30
46830 / 1kbp
54Hold
Cycle: sequence 2~4 × (25~45)

STEP DOWN

SequenceTemp. (°C)Time (sec)
194120
29810
37430 / 1kbp
49810
57230
69810
77030
89810
96830
1068420
114Hold
Cycle:
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

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
SequenceTemp. (°C)Time (min)
137120
26515
34Hold

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.
SolutionVector DNAInsert DNADWMighty MixTotal
Volume (µL)122510
Thermal protocol is following
SequenceTemp. (°C)Time (min)
11630
26510
34Hold

Electrophoresis

  1. Put gel into electrophoresis tank.
  2. Pour 2x TBE buffer into the tank to soak gel.
  3. Add 5 µL of EtBr into cathode.
  4. Pre-migration for 30 min at 100 V.
  5. Apply DNA solutions with 6x loading dye and ladder.
  6. Start electrophoresis at 100 V.
  7. Stop at appropriate time.

Gel Extraction

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Ethanol precipitation

  1. Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
  2. Leave it at room temperature for 5 min.
  3. Centrifuge at 15,000 rpm for 15 min at 25°C.
  4. Remove supernatant and add 600 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 5 min at 25°C.
  6. Remove supernatant and dry up at room temperature with light sheilding.
  7. 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
SequenceTemp. (°C)Time (sec)
194120
29430
36860 / 1kbp
44Hold
Cycles: sequence 2~3 × 25~45

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
SequenceTemp. (°C)Time (sec)
19610
2505
360240
44Hold
Cycle: sequence 2~4 × 25

Ethanol precipitation

Solution PCR product DW 3M NaOAc Glycogen 100% EtOH
Volume (µL) 10 10 2 1 50
  1. Centrifuge at 15,000 rpm for 15 min at room temprature.
  2. Remove supernatant, add 100 µL of 70% EtOH and tap tubes by finger.
  3. Centrifuge at 15,000 rpm for 10 min at room temprature.
  4. Remove supernatant and dry up at room temperature.
  5. Resuspend the pellet to HiDi formamide and remove to 96-well plate.
  6. Set the plate and start electrophoresis.

Competent Cells

  1. Thaw original competent cells on ice.
  2. Add 5 µL of original competent cells to 2 mL of LB.
  3. Incubate the cells for 16 hrs at 37°C.
  4. Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
  5. Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
  6. Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
  7. Remove supernatant and add 75 mL of TB to each tube.
  8. Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
  9. Remove supernatant and add 32 mL of TB.
  10. Add 32 µL of DMSO 10 times.
  11. Take 50 µL and freeze with liquid nitrogen.

Transformation

  1. Add plasmid to thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add to LB.
  4. Incubate the cells for 2 hrs at 37°C.
  5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
  6. 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

Streaking (Single colony isolation)

  1. Pick the colony with an inoculating loop from the agar plate．
  2. Drag the loop across on a new agar plate.
  3. Resterilize the loop and drag it across again.
  4. Repeat method 3.

PEG precipitation

  1. Add 13 µL of PEG to 20 µL of product(s).
  2. Leave it at room temperature for 1 hr.
  3. Centrifuge at 15,000 rpm for 20 min at 4°C.
  4. Remove supernatant and add 100 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 2 min at 4°C.
  6. Remove supernatant and dry up at room temperature with light sheilding.
  7. 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
Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
36830 / 1kbp
44Hold
Cycle: sequence 2~3 × (25~45)

Preparation of magnetic beads

  1. Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
  2. Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
  3. Remove supernatant.

Fixation to magnetic beads

  1. Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
  2. Mix by vibration using sonic-toothbrush.
  3. Collect the beads using magnet.
  4. Remove supernatant containing excess amount of free DNA fragment.

Double restriction digestion

  1. 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.
  2. Mix by pumping using pipette.
  3. Incubate at 37 °C for 30 min.
  4. Collect the beads using magnet.
  5. Obtain supernatant containing digested DNA fragment.
  6. Purify the supernatant by ethanol precipitation.