Team:Groningen/protocols/PCR

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<h2>PCR</h2>

<h5>Materials:</h5>
<ul type="square">
<li>PCR tubes</li>
<li>MQ water</li>
<li>DMSO 5%</li>
<li>Phusion buffer</li>
<li>DNTPs</li>
<li>F- and R-primer</li>
<li>Phusion Polymerase</li>
<li>DNA template</li>
</ul>

<p>
<br>
<h5>PCR reaction mixture:</h5>
<table border="1"  >
  <tr width="15%" align="center" >
    <th >Component</th>
    <th >50 &micro;l</th>
    <th >Final concentration</th>
  </tr>

  <tr>
    <td><ul>MQ water</td>
    <td><ul>up to 50 &micro;l</td>
    <td><ul></td>
  </tr>

  <tr>
    <td><ul>5x Phusion Buffer</td>
    <td><ul>10 &micro;l</td>
    <td><ul>1x</td>
  </tr>

  <tr>
    <td><ul>DMSO 5%</td>
    <td><ul>1.5 &micro;l</td>
    <td><ul></td>
  </tr>

  <tr>
    <td><ul>10 mM dNTPs</td>
    <td><ul>1&micro;l</td>
    <td><ul>200 &micro;M</td>
  </tr>

  <tr>
    <td><ul>primer F</td>
    <td><ul>2.5 &micro;l</td>
    <td><ul>0.5 &micro;M</td>
  </tr>

  <tr>
    <td><ul>primer R</td>
    <td><ul>2.5 &micro;l</td>
    <td><ul>0.5 &micro;M</td>
  </tr>

  <tr>
    <td><ul>template DNA</td>
    <td><ul>1 &micro;l</td>
    <td><ul>&sim;1 ng</td>
  </tr>

   <tr>
    <td><ul>Phusion Polymerase <br>2 U/&micro;l</td>
    <td><ul>0.3 &micro;l</td>
    <td><ul>0.01 U/&micro;l</td>
  </tr>

</table> 

<p>
<br>
<h5>Cycling instructions:</h5>
<table border="1">
  <tr>
    <th align=center width="100">Temperature</th>
    <th align=center width="100">Time</th>
    <th align=center width="100">Number of Cycles</th>
  </tr>

  <tr>
    <td><ul>98&deg;C</td>
    <td><ul>2 min</td>
    <td><ul>1</td>
  </tr>

  <tr>
    <td><ul>98&deg;C</td>
    <td><ul>10 sec</td>
    <td rowspan="3"><ul>34</td>
  </tr>

  <tr>
    <td><ul>T<sub>m</sub>-5&deg;C</td>
    <td><ul>25 sec</td>
  </tr>

  <tr>
    <td><ul>72&deg;C</td>
    <td><ul>30 sec/kb</td>
  </tr>

  <tr>
    <td><ul>72&deg;C</td>
    <td><ul>10 min</td>
    <td><ul>1</td>
  </tr>

  <tr>
    <td><ul>4&deg;C</td>
    <td><ul>&infin;</td>
    <td><ul>1</td>
  </tr>
</table> 

<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf

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