Team:Washington/GENERAL PCR PROTOCOL

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<h1 dir="ltr">General PCR Protocol</h1>
<p dir="ltr">See also: <a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">PCR_GoTaq</a>
<br />
<p dir="ltr">Resuspending primers:</p>
<p dir="ltr"><a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">Protocol: Resuspending PCR primers</a></p>
<br />
<p dir="ltr">Working Stock: </p>
<p dir="ltr">(serves as a backup)</p>
<p dir="ltr">Dilute to 10 times the number on primer vial</p>
<p dir="ltr">10ul fwd primer 90ul DI water</p>
<p dir="ltr">10ul rev primer 90ul DI water</p>
<br />
<p dir="ltr">Notes for iGEM 2013 HSB lab:</p>
<p dir="ltr">We are using the GreenTaq polymerase 2X pre-mixed (<a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>) for PCR. </p>
<p dir="ltr">Use the protocols below for Green Taq polymerase. </p>
<p dir="ltr">Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).</p>
<p dir="ltr">If PCRing insert use 1ng/ul DNA concentration.</p>
<br />
<p dir="ltr">Amplification PCR with Green Taq polymerase:</p>
<ol>
  <li dir="ltr">
    <p dir="ltr">Setup Amplification PCR Reaction</p>
  </li>
  <ol>
    <li dir="ltr">
      <p dir="ltr">1uL Template DNA</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">22ul deionized (dI) H2O</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1ul 10uM forward primer (Tm XX C)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1ul 10uM reverse Primer (Tm YY C)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">25ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">Total 50ul</p>
    </li>
  </ol>
  <li dir="ltr">
    <p dir="ltr">Amplification PCR Reaction Program</p>
  </li>
  <ol>
    <ol>
      <ol>
        <li dir="ltr">
          <p dir="ltr">95 C for 30s</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">95 C for 30s</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">ZZ* C for 30s (important step)</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">72 C for 1min per kb target gene plus 10-15% (important step) </p>
        </li>
        <li dir="ltr">
          <p dir="ltr">Repeat 2-4 34x</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">72 C for 5 to 10min</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">4 C for forever (don&rsquo;t forget to turn the machine off the next day)</p>
        </li>
      </ol>
    </ol>
  </ol>
</ol>
<ul>
  <li dir="ltr">
    <p dir="ltr">Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn&rsquo;t have to be exact (within 5 degrees or so).</p>
  </li>
</ul>
<br />
<p dir="ltr">Analytical PCR with Green Taq polymerase:</p>
<ol start="3">
  <li dir="ltr">
    <p dir="ltr">Setup PCR Reaction</p>
  </li>
  <ol>
    <li dir="ltr">
      <p dir="ltr">1uL Template</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">2ul dI H2O</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1ul 10uM forward primer (Tm XX C)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1ul 10uM reverse Primer (Tm YY C)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">5ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">Total 10uls</p>
    </li>
  </ol>
  <li dir="ltr">
    <p dir="ltr">PCR Reaction Program</p>
  </li>
  <ol>
    <ol>
      <ol>
        <li dir="ltr">
          <p dir="ltr">95 C - 30s</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">95 C - 10s</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">ZZ* C - 10s (important step)</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">72 C - 1min per kb target gene plus 10-15% (important step) </p>
        </li>
        <li dir="ltr">
          <p dir="ltr">Repeat b-d 25x</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">72 C - 5 to 10min</p>
        </li>
        <li dir="ltr">
          <p dir="ltr">10 or 4 C - forever</p>
        </li>
      </ol>
    </ol>
  </ol>
</ol>
<br />
<p dir="ltr">Phusion polymerase:</p>
<ol start="5">
  <li dir="ltr">
    <p dir="ltr">Setup Amplification PCR Reaction</p>
  </li>
  <ol>
    <li dir="ltr">
      <p dir="ltr">1uL Template</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1uL 25mM dNTP's</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">10uL Phusion HF Buffer</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1.0ul Forward Primer (Tm XX oC)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">1.0ul Reverse Primer (Tm YY oC)*</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">0.5uL Phusion polymerase</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">36.5uL diH2O</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">Total 50uls</p>
    </li>
  </ol>
  <li dir="ltr">
    <p dir="ltr">Amplification PCR Reaction Program</p>
  </li>
  <ol>
    <li dir="ltr">
      <p dir="ltr">98 C - 30s</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">98 C - 10s</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">ZZ* C - 10s (important step)</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">72 C - 30s per kb target gene plus 10-15% (important step) </p>
    </li>
    <li dir="ltr">
      <p dir="ltr">Repeat b-d 29-34x</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">72 C - 5 to 10min</p>
    </li>
    <li dir="ltr">
      <p dir="ltr">10 or 4 C - forever</p>
    </li>
  </ol>
</ol>
<ul>
  <li dir="ltr">
    <p dir="ltr">Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn&rsquo;t have to be exact (within 5 degrees or so).</p>
  </li>
</ul>
<p dir="ltr">Refer for further details to NEB's online protocol for Phusion:</p>
<p dir="ltr"><a href="http://www.neb.com/nebecomm/products/protocol87.asp">Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase</a</p>
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