Difference between revisions of "Team:Paris Bettencourt/Protocols/PCR"

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     <td><b>PCR protocol (Phusion)</b><br />
 
     <td><b>PCR protocol (Phusion)</b><br />
 
     <ul>
 
     <ul>
−
       <li>Thaw PCR Master Mix on ice for 10min.
+
       <li>Prepare the following mix
−
      <li>add 2µL of each primers (at 10 µM) in a PCR tube.
+
        <ul>
−
      <li>add 1µL of the DNA (at 1 ng/µL) you want to amplify in the same tube.
+
          <li>2µL primer Forward (10 µM)</li>
−
      <li>add 25µL of <i>Life Technologies</i> Phusion High-Fidelity PCR Master Mix with HF Buffer.
+
          <li>2µL primer Reverse (10 µM)</li>
−
      <li>add 20µL of DNase/RNase-free water to the tube.
+
          <li>0.1 to 1 ng of template DNA</li>
−
       <li>Thermocycling conditions
+
          <li>up to 50µL of DNase/RNase-free water</li>
−
           <ul>
+
          <li>25µL of <i>Life Technologies</i> Phusion High-Fidelity PCR Master Mix with HF Buffer</li>
−
          <li> initial denaturation 98°C for 30 seconds
+
        </ul>
−
          <li>35 cycles
+
       <li>Launch the PCR using the following mix
−
              <ul>
+
 
−
              <li>98°C for 30 seconds
+
           <table style="width:25%">
−
              <li>57°C for 30 seconds
+
 
−
              <li> 72°C 30 seconds/kb
+
  <tr>
−
              </ul>
+
    <th>time (min)</th>
−
          <li> final extension of 72°C for 10 minutes
+
    <th>temperature (°C)</th>
−
          <li> infinite hold 12°C
+
    <th>function</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>3:00</td>
 +
    <td>98</td>
 +
    <td>melting</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td> </td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>0:30</td>
 +
    <td>98</td>
 +
    <td>melting</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>0:30</td>
 +
    <td>64</td>
 +
    <td>annealing</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>1:00</td>
 +
    <td>72</td>
 +
    <td>extension</td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
  </tr>
 +
  <tr>
 +
    <td>10:00</td>
 +
    <td>72</td>
 +
    <td>extension</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>forever</td>
 +
    <td>12</td>
 +
    <td>storage</td>
 +
  </tr>
 +
</table>
 
           </ul>
 
           </ul>
 
     </ul>
 
     </ul>
 
     </td>
 
     </td>
 
   </tr>
 
   </tr>
−
</table>
+
</table></li>
  
 
</html>
 
</html>
 
{{Paris_Bettencourt/footer}}
 
{{Paris_Bettencourt/footer}}

Revision as of 16:00, 1 September 2015

PCR protocol (Phusion)
  • Prepare the following mix
    • 2µL primer Forward (10 µM)
    • 2µL primer Reverse (10 µM)
    • 0.1 to 1 ng of template DNA
    • up to 50µL of DNase/RNase-free water
    • 25µL of Life Technologies Phusion High-Fidelity PCR Master Mix with HF Buffer
  • Launch the PCR using the following mix
    time (min) temperature (°C) function
    3:00 98 melting
    0:30 98 melting
    0:30 64 annealing
    1:00 72 extension
    10:00 72 extension
    forever 12 storage