Team:Evry/Protocols

From 2012.igem.org

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<h1>PCR with Phusion polymerase<h1>
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<h1>PCR with Phusion High-Fidelity DNA Polymerase</h1>
 +
 
 +
<h2>Tube preparation</h2>
 +
Put items in this order:
 +
 
 +
<table>
 +
      <tr>
 +
            <td>Component</td>
 +
            <td>50µl reaction</td>
 +
            <td>Comments</td>
 +
      </tr>
 +
      <tr>
 +
            <td>H2O</td>
 +
            <td>32</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>5x Phusion HF Buffer</td>
 +
            <td>10</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>10mM dNTPs</td>
 +
            <td>1</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>Primer FW</td>
 +
            <td>2</td>
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            <td>Primers have to be at 10µM</td>
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      </tr>
 +
      <tr>
 +
            <td>Primer RV</td>
 +
            <td>2</td>
 +
            <td>Primers have to be at 10µM</td>
 +
      </tr>
 +
      <tr>
 +
            <td>Template DNA</td>
 +
            <td>1</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>DMSO (optional)</td>
 +
            <td>1,5</td>
 +
            <td>recommended for GC-rich amplicons < 20kb</td>
 +
      </tr>
 +
      <tr>
 +
            <td>Phusion DNA polymerase</td>
 +
            <td>0,5</td>
 +
            <td></td>
 +
      </tr>
 +
</table>
 +
 
 +
<h2>Cycling instructions</h2>
 +
 
 +
<table>
 +
      <tr>
 +
          <td>Cycle step</td>
 +
          <td>Temperature</td>
 +
          <td>Time</td>
 +
          <td>Cycles</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Initial denaturation</td>
 +
          <td>98°C</td>
 +
          <td>4min</td>
 +
          <td>1</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Denaturation</td>
 +
          <td>98°C</td>
 +
          <td>20s</td>
 +
          <td rowspan="3">30</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Annealing</td>
 +
          <td>Lower Tm of primers</td>
 +
          <td>30s</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Extension</td>
 +
          <td>72°C</td>
 +
          <td>30S/kb</td>
 +
      </tr>
 +
      <tr>
 +
          <td rowspan="2">Final extension</td>
 +
          <td>72°C</td>
 +
          <td>10min</td>
 +
          <td rowspan="1">1</td>
 +
      </tr>
 +
      <tr>
 +
          <td>4°C</td>
 +
          <td>hold</td>
 +
      </tr>
 +
 
<h1>Préparation of LB medium and LB Agar:</h1>
<h1>Préparation of LB medium and LB Agar:</h1>

Revision as of 12:59, 3 August 2012

Contents

PCR with Phusion High-Fidelity DNA Polymerase

Tube preparation

Put items in this order:

Component 50µl reaction Comments
H2O 32
5x Phusion HF Buffer 10
10mM dNTPs 1
Primer FW 2 Primers have to be at 10µM
Primer RV 2 Primers have to be at 10µM
Template DNA 1
DMSO (optional) 1,5 recommended for GC-rich amplicons < 20kb
Phusion DNA polymerase 0,5

Cycling instructions

Préparation of LB medium and LB Agar:


=> LB Agar : -18,5g LB Agar -300ml H2O

=> LB medium : -6g LB broth -300ml de H2O

Autoclaved at 250°C














Cycle step Temperature Time Cycles
Initial denaturation 98°C 4min 1
Denaturation 98°C 20s 30
Annealing Lower Tm of primers 30s
Extension 72°C 30S/kb
Final extension 72°C 10min 1
4°C hold
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