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

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<html>
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Protocol
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<a href="https://2016.igem.org/Team:HokkaidoU_Japan/Notebook/notebook">notebook</a>
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<br>
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<h1>Protocols</h1>
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<a href=https://2016.igem.org/Team:HokkaidoU_Japan/Notebook/notebook>to dokodoko</a>
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<br>
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<h2 onClick="hyoji1()" style="cursor:hand; cursor:pointer">PCR</h2>
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<div id="disp1">
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<p>
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  Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
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</p>
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<table>
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  <tr>
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    <th>Solution</th>
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    <td>template DNA</td>
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    <td>Primer-F 10&micro;M</td>
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    <td>Primer-R 10&micro;M</td>
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    <td>MgSO<sub>4</sub></td>
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    <td>dNTPs</td>
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    <td>10x Buffer</td>
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    <td>KOD Plus Neo</td>
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    <td>DW</td>
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    <td>Total</td>
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  </tr>
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  <tr>
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    <th>Volume (&micro;L)</th>
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    <td>1</td>
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    <td>1</td>
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    <td>1</td>
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    <td>3</td>
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    <td>5</td>
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    <td>5</td>
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    <td>1</td>
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    <td>33</td>
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    <td>50</td>
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  </tr>
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</table>
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<p>Thermal protocol is following</p>
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<h3>2STEP Cycle (Tm value &gt; 63&deg;C)</h3>
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<table>
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  <tr>
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    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
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  </tr>
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  <tr>
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    <td>1</td><td>94</td><td>120</td>
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  </tr>
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  <tr>
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    <td>2</td><td>98</td><td>10</td>
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  </tr>
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  <tr>
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    <td>3</td><td>68</td><td>30sec / 1kbp</td>
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  </tr>
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  <tr>
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    <td>4</td><td>4</td><td>Hold</td>
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  </tr>
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</table>
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<p>Cycle: sequence2~3 &times; (25~45)</p>
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<h3>3STEP Cycle (Tm value &lt; 63&deg;C)</h3>
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<table>
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  <tr>
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    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
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  </tr>
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  <tr>
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    <td>1</td><td>94</td><td>120</td>
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  </tr>
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  <tr>
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    <td>2</td><td>98</td><td>10</td>
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−
  </tr>
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−
  <tr>
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    <td>3</td><td>Tm</td><td>30</td>
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  </tr>
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  <tr>
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    <td>4</td><td>68</td><td>30sec / 1kbp</td>
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  </tr>
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  <tr>
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    <td>5</td><td>4</td><td>Hold</td>
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  </tr>
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</table>
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<p>Cycle: sequence2~4 &times; (25~45)</p>
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</div>
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<script>
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document.getElementById("disp1").style.display="none";
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var displaying=0;
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function hyoji1()
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{
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  if (displaying==0)
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  {
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    document.getElementById("disp1").style.display="block";
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displaying=1;
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  }
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  else
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  {
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    document.getElementById("disp1").style.display="none";
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displaying=0;
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−
  }
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−
}
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</script>
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−
 
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−
 
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−
 
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<h2 onClick="hyoji2()" style="cursor:hand; cursor:pointer">PCR Purification</h2>
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<div id="disp2">
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<p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
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<br>Purification of PCR products</p>
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</div>
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<script>
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document.getElementById("disp2").style.display="none";
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var displaying=0;
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function hyoji2()
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{
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  if (displaying==0)
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  {
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    document.getElementById("disp2").style.display="block";
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displaying=1;
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  }
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  else
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  {
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−
    document.getElementById("disp2").style.display="none";
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displaying=0;
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−
  }
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−
}
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</script>
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−
 
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<h2 onClick="hyoji3()" style="cursor:hand; cursor:pointer">Digestion</h2>
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<div id="disp3">
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<p>Mix the following reagents in PCR tube.</p>
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<table>
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  <tr>
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    <th>Solution</th>
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    <td>DNA</td>
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    <td>RE1 10U/&micro;L</td>
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    <td>RE2 10U/&micro;L</td>
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    <td>Appropriate buffer</td>
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    <td>Total</td>
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  </tr>
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  <tr>
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    <th>Volume (&micro;L)</th>
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    <td>16</td>
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    <td>1</td>
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    <td>1</td>
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    <td>2</td>
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    <td>20</td>
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  </tr>
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</table>
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−
<table>
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−
  <tr>
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−
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
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  </tr>
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−
  <tr>
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−
    <td>1</td><td>37</td><td>120</td>
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  </tr>
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  <tr>
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    <td>2</td><td>65</td><td>15</td>
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  </tr>
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  <tr>
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    <td>3</td><td>4</td><td>Hold</td>
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−
  </tr>
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−
</table>
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−
 
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−
</div>
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−
<script>
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−
document.getElementById("disp3").style.display="none";
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−
var displaying=0;
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−
function hyoji3()
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−
{
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−
  if (displaying==0)
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−
  {
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−
    document.getElementById("disp3").style.display="block";
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−
displaying=1;
+
−
  }
+
−
  else
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−
  {
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−
    document.getElementById("disp3").style.display="none";
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−
displaying=0;
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−
  }
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−
}
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−
</script>
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−
 
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<h2 onClick="hyoji4()" style="cursor:hand; cursor:pointer">Ligation</h2>
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<div id="disp4">
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<p>
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  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.
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</p>
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<table>
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−
  <tr>
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    <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td>
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−
  </tr>
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−
  <tr>
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−
    <th>Volume (&micro;L)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td>
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−
  </tr>
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−
</table>
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−
<p>Thermal protocol is following</p>
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−
<table
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−
  <tr>
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−
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
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−
  </tr>
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−
  <tr>
+
−
    <td>1</td><td>16</td><td>30</td>
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−
  </tr>
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−
  <tr>
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−
    <td>2</td><td>65</td><td>10</td>
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−
  </tr>
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−
  <tr>
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−
    <td>3</td><td>4</td><td>Hold</td>
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−
  </tr>
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−
</table>
+
−
 
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−
</div>
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−
<script>
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−
document.getElementById("disp4").style.display="none";
+
−
var displaying=0;
+
−
function hyoji4()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp4").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp4").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
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−
 
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−
<h2 onClick="hyoji5()" style="cursor:hand; cursor:pointer">Electrophoresis</h2>
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−
<div id="disp5">
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−
<ol>
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−
  <li>Put gel into electrophoresis tank.</li>
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−
  <li>Pore 2x TBE buffer into the tank to soak gel.</li>
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−
  <li>Add 5  &micro;L  of EtBr into cathod.</li>
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−
  <li>Pre-migration for 30 min at 100 V.</li>
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−
  <li>Apply DNA solution with 6x loading dye and ladder.</li>
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  <li>Start electrophoresis at 100 V.</li>
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−
</ol>
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−
 
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−
</div>
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−
<script>
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−
document.getElementById("disp5").style.display="none";
+
−
var displaying=0;
+
−
function hyoji5()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp5").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp5").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
+
−
 
+
−
 
+
−
<h2 onClick="hyoji6()" style="cursor:hand; cursor:pointer">Gel Extraction</h2>
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−
<div id="disp6">
+
−
 
+
−
<p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
+
−
<br>DNA extraction from gel</p>
+
−
 
+
−
</div>
+
−
<script>
+
−
document.getElementById("disp6").style.display="none";
+
−
var displaying=0;
+
−
function hyoji6()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp6").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp6").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
+
−
 
+
−
 
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−
<h2 onClick="hyoji7()" style="cursor:hand; cursor:pointer">Ethanol precipitation</h2>
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−
<div id="disp7">
+
−
<ol>
+
−
  <li>Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.</li>
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−
  <li>Leave it at room temperature for 5 min.</li>
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−
  <li>Centrifuge at 15,000 rpm for 15 min at 25&deg;C.</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>Remove supernatant and air-dry at room temperature with light sheilding.</li>
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−
  <li>Suspend with 10  &micro;L  of TE.</li>
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−
</ol>
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−
 
+
−
 
+
−
</div>
+
−
<script>
+
−
document.getElementById("disp7").style.display="none";
+
−
var displaying=0;
+
−
function hyoji7()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp7").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp7").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
+
−
 
+
−
<h2 onClick="hyoji8()" style="cursor:hand; cursor:pointer">Colony PCR</h2>
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−
<div id="disp8">
+
−
<table>
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−
  <tr>
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−
    <th>Solution</th>
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−
    <td>DNA</td>
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−
    <td>Kapa-Taq (Taq polymerase)</td>
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−
    <td>EX-F primer 10&micro;M</td>
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−
    <td>PS-R primer 10&micro;M</td>
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−
    <td>Total</td>
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−
  </tr>
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−
  <tr>
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−
    <th>Volume (&micro;L)</th>
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−
    <td>4.2</td>
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−
    <td>5</td>
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−
    <td>0.4</td>
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−
    <td>0.4</td>
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−
    <td>10</td>
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−
  </tr>
+
−
</table>
+
−
 
+
−
<table>
+
−
  <tr>
+
−
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>1</td><td>94</td><td>120</td>
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−
  </tr>
+
−
  <tr>
+
−
    <td>2</td><td>94</td><td>30</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>3</td><td>68</td><td>60 sec / 1kbp</td>
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−
  </tr>
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−
    <td>4</td><td>4</td><td>Hold</td>
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−
  </tr>
+
−
</table>
+
−
<p>Cycles: sequence2~3 &times; 25~45</p>
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−
 
+
−
 
+
−
</div>
+
−
<script>
+
−
document.getElementById("disp8").style.display="none";
+
−
var displaying=0;
+
−
function hyoji8()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp8").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp8").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
+
−
 
+
−
<h2 onClick="hyoji9()" style="cursor:hand; cursor:pointer">Sequencing</h2>
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−
<div id="disp9">
+
−
<table>
+
−
  <tr>
+
−
    <th>Solution</th>
+
−
    <td>5 x Sequencing Buffer</td>
+
−
    <td>primer 1&micro;M</td>
+
−
    <td>template DNA</td>
+
−
    <td>Ready Reaction Premix</td>
+
−
    <td>DW</td>
+
−
    <td>Total</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <th>Volume (&micro;L)</th>
+
−
    <td>1.5</td>
+
−
    <td>1.5</td>
+
−
    <td>1</td>
+
−
    <td>1</td>
+
−
    <td>5</td>
+
−
    <td>10</td>
+
−
  </tr>
+
−
</table>
+
−
 
+
−
<table>
+
−
  <tr>
+
−
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>1</td><td>96</td><td>10</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>2</td><td>50</td><td>5</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>3</td><td>60</td><td>240</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>4</td><td>4</td><td>Hold</td>
+
−
  </tr>
+
−
</table>
+
−
<p>Cycle: sequence2~4 &times; 25</p>
+
−
 
+
−
 
+
−
<h3>Ethanol precipitation</h3>
+
−
<table>
+
−
  <tr>
+
−
    <th>Solution</th>
+
−
    <td>PCR product</td>
+
−
    <td>DW</td>
+
−
    <td>3M NaOAc</td>
+
−
    <td>Glycogen</td>
+
−
    <td>100% EtOH</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <th>Volume (&micro;L)</th>
+
−
    <td>10</td>
+
−
    <td>10</td>
+
−
    <td>2</td>
+
−
    <td>1</td>
+
−
    <td>50</td>
+
−
  </tr>
+
−
</table>
+
−
<ol>
+
−
  <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>Centrifuge at 15,000 rpm for 10 min at room temprature</li>
+
−
  <li>Remove supernatant and air dry at room temperature.</li>
+
−
  <li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li>
+
−
  <li>Set the plate and start electrophoresis.</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<script>
+
−
document.getElementById("disp9").style.display="none";
+
−
var displaying=0;
+
−
function hyoji9()
+
−
{
+
−
  if (displaying==0)
+
−
  {
+
−
    document.getElementById("disp9").style.display="block";
+
−
displaying=1;
+
−
  }
+
−
  else
+
−
  {
+
−
    document.getElementById("disp9").style.display="none";
+
−
displaying=0;
+
−
  }
+
−
}
+
−
</script>
+
−
 
+
−
 
+
−
<h2 onClick="hyoji10()" style="cursor:hand; cursor:pointer">Competent Cells</h2>
+
−
<div id="disp10">
+
−
<ol>
+
−
<li>Thaw original competent cells on ice.</li>
+
−
<li>Add 5 &micro;L of original competent cells to 2 mL of LB.</li>
+
−
<li>Incubate the cells for 16 hrs at 37&deg;C.</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>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>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>Remove supernatant and add 32 mL of TB.</li>
+
−
<li>Add 32 &micro;L of DMSO 10 times.</li>
+
−
<li>Take 50 &micro;L and freeze with liquid nitrogen.</li>
+
−
</ol>
+
−
 
+
−
</div>
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</script>
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<h2 onClick="hyoji11()" style="cursor:hand; cursor:pointer">Transformation</h2>
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<div id="disp11">
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<ol>
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  <li>Add plasmid to thawed competent cells on ice.</li>
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  <li>Incubate on ice for 30 min.</li>
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  <li>Add to LB.</li>
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  <li>(Incubate the cells for 2 hrs at 37&deg;C.)</li>
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  <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
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  <li>Incubate the plate(s) at 37&deg;C for 16~20 hrs.</li>
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</ol>
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  }
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}
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</script>
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<h2 onClick="hyoji12()" style="cursor:hand; cursor:pointer">Mini-prep</h2>
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<div id="disp12">
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<p>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd)
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<br>fast / standard / low copy protocol</p>
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</div>
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  }
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}
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</script>
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<h2 onClick="hyoji13()" style="cursor:hand; cursor:pointer">Streaking (Single colony isolation)</h2>
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<div id="disp13">
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<ol>
+
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  <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>Re-sterilise the loop and drag it across again.</li>
+
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  <li>Repeat method 3.</li>
+
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</ol>
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</div>
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displaying=0;
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  }
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}
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</script>
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<h2 onClick="hyoji14()" style="cursor:hand; cursor:pointer">PEG precipitation</h2>
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<div id="disp14">
+
−
<ol>
+
−
  <li>Add 13  &micro;L  of PEG to 20  &micro;L  of product(s).</li>
+
−
  <li>Leave it at room temperature for 1 hr.</li>
+
−
  <li>Centrifuge at 15,000 rpm for 20 min at 4&deg;C.</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>Remove supernatant and air-dry at room temperature with light sheilding.</li>
+
−
  <li>Suspend with 10  &micro;L  of TE.</li>
+
−
</ol>
+
−
 
+
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</div>
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<script>
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  }
+
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}
+
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</script>
+
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+
−
<h2 onClick="hyoji15()" style="cursor:hand; cursor:pointer">Gel Free System</h2>
+
−
<div id="disp15">
+
−
<h3>Preparation of biotinylated DNA fragments</h3>
+
−
<p>
+
−
  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>
+
−
  <tr>
+
−
    <th>Solution</th>
+
−
    <td>template DNA</td>
+
−
    <td>5'-biotinylated 100-UP primer 10&micro;M</td>
+
−
    <td>5'-biotinylated 200-DN primer 10&micro;M</td>
+
−
    <td>MgSO<sub>4</sub></td>
+
−
    <td>dNTPs</td>
+
−
    <td>10x Buffer</td>
+
−
    <td>KOD Plus Neo</td>
+
−
    <td>DW</td>
+
−
    <td>Total</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <th>Volume (&micro;L)</th>
+
−
    <td>1</td>
+
−
    <td>1.5</td>
+
−
    <td>1.5</td>
+
−
    <td>3</td>
+
−
    <td>5</td>
+
−
    <td>5</td>
+
−
    <td>1</td>
+
−
    <td>32</td>
+
−
    <td>50</td>
+
−
  </tr>
+
−
</table>
+
−
<p>Thermal protocol is following</p>
+
−
<h3>2STEP Cycle (Tm value &gt; 63&deg;C)</h3>
+
−
<table>
+
−
  <tr>
+
−
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>1</td><td>94</td><td>120</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>2</td><td>98</td><td>10</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>3</td><td>68</td><td>30sec / 1kbp</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>4</td><td>4</td><td>Hold</td>
+
−
  </tr>
+
−
</table>
+
−
<p>Cycle: sequence2~3 &times; (25~45)</p>
+
−
 
+
−
<h3>Preparation of magnetic beads</h3>
+
−
<ol>
+
−
  <li>Mix 2 &micro;L  of magnetic beads (SiMAG-Streptavidin) and 48 &micro;L  of TE by vibration using sonic-toothbrush.</li>
+
−
  <li>Collect the beads by attracting them to one side in 0.2 mL  polypropylene tube using neodymium magnet.</li>
+
−
  <li>Remove supernatant.</li>
+
−
</ol>
+
−
 
+
−
<h3>Fixation to magnetic beads</h3>
+
−
<ol>
+
−
  <li>Add 3 &micro;L  of PCR product (0.48 pmol) and 7 &micro;L  of TE to beads.</li>
+
−
  <li>Mix by vibration using sonic-toothbrush.</li>
+
−
  <li>Collect the beads using magnet.</li>
+
−
  <li>Remove supernatant containing excess amount of free DNA fragment.</li>
+
−
</ol>
+
−
 
+
−
<h3>Double restriction digestion</h3>
+
−
<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>Mix by pumping using pipette.</li>
+
−
  <li>Incubate at 37 &deg;C for 30 min.</li>
+
−
  <li>Collect the beads using magnet.</li>
+
−
  <li>Obtain supernatant containing digested DNA fragment.</li>
+
−
  <li>Purify the supernatant by ethanol precipitation.</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<script>
+
−
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+
−
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+
−
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+
−
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+
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+
−
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+
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−
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+
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+
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  else
+
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+
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+
−
  }
+
−
}
+
−
</script>
+
−
 
+
 
</html>
 
</html>

Revision as of 05:31, 24 August 2016

Protocol notebook