Difference between revisions of "Team:UiOslo Norway/Experiments/PCR phusion"

 
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<p>
 
<p>
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Using the guidelines of the provided Phusion® High-Fidelity DNA Polymerase (M0530)
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<a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530" >
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protocol.
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</a>
 
<ol>
 
<ol>
 
   <li><p><b>Reaction Setup:</b></br>
 
   <li><p><b>Reaction Setup:</b></br>
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   <tr>
 
   <tr>
 
     <td>Initial Denaturation</td>
 
     <td>Initial Denaturation</td>
−
     <td>95 °C</td>
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     <td>98 °C</td>
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     <td>30 seconds /5 Minutes for Colony PCR
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     <td>30 seconds</td>
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  </td>
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   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
 
     <td rowspan="3"></br></br>35 Cycles</td>
 
     <td rowspan="3"></br></br>35 Cycles</td>
−
     <td>95 °C</td>
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     <td>98 °C</td>
−
     <td>30 seconds</td>
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     <td>10 seconds</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
     <td>45 °C – 72 °C</td>
 
     <td>45 °C – 72 °C</td>
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     <td>60 seconds</td>
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     <td>30 seconds</td>
 
   </tr>
 
   </tr>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
     <td>68 °C</td>
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     <td>72 °C</td>
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     <td>1 minute / 1 kb</td>
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     <td>30 seconds / 1 kb</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
 
     <td>Final Extension</td>
 
     <td>Final Extension</td>
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     <td>68 °C</td>
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     <td>72 °C</td>
 
     <td>5 minutes</td>
 
     <td>5 minutes</td>
 
   </tr>
 
   </tr>
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<div class="clear"></div>
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<!---------------sponsors------------------------->
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<center><h1>iGEM UiOslo 2015 is sponsored by:</h1>
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<hr style="height:2px;border:none;color:black;background-color:black;" />
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<img src="https://static.igem.org/mediawiki/2015/a/ac/UiOslo_Statoil.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/e/e0/UiOslo_uio_logo.png" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/0/08/UiOslo_Enova1.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/e/eb/UiOslo_GATC-Biotech.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/4/4a/UiOslo_IDT.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/d/d9/UiOslo_Norwegian_Biochemical_Society.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/f/f0/UiOslo_SnapGene.jpg" Height="60px" hspace="15">
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</center>
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<hr style="height:2px;border:none;color:black;background-color:black;" />
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<!--------end sponsors---------------------------->
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</div>
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<!--------end sponsors---------------------------->
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</body>
 
</html>
 
</html>

Latest revision as of 12:09, 18 September 2015

PCR with Phusion-HF DNA Polymerase (NEB):

Back to Protocols


Using the guidelines of the provided Phusion® High-Fidelity DNA Polymerase (M0530) protocol.

  1. Reaction Setup:

    10 µl 5X Phusion HF Buffer
    5 µl dNTPS [2 mM]
    2.5 µl Forward Primer [10 µM]
    2.5 µl Reverse Primer [10 µM]
    1 µl Template DNA
    0.5 µl Phusion HF DNA Polymerase
    28.5 µl MilliQ Water

    50 µl Total Reaction


  2. PCR Program:

    Step Temperature Time
    Initial Denaturation 98 °C 30 seconds


    35 Cycles    		 98 °C 10 seconds
    45 °C – 72 °C 30 seconds
    72 °C 30 seconds / 1 kb
    Final Extension 72 °C 5 minutes
    Hold 4 °C forever

    3. iGEM UiOslo 2015 is sponsored by: