Team:Groningen/protocols/PCR

From 2013.igem.org

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<table id="normal" width="60%">
<table id="normal" width="60%">
   <tr>
   <tr>
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     <th><ul>Component</th>
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     <th>Component</th>
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     <th><ul>50 ml</th>
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     <th>50 ml</th>
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     <th><ul>Final concentration</th>  
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     <th>Final concentration</th>  
   </tr>
   </tr>
   <tr>
   <tr>
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     <td><ul>MQ water</td>
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     <td>MQ water</td>
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     <td><ul>up to 50 &micro;l</td>
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     <td>up to 50 &micro;l</td>
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     <td><ul></td>
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     <td></td>
   </tr>
   </tr>
   <tr>
   <tr>
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     <td><ul>5x Phusion Buffer</td>
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     <td>5x Phusion Buffer</td>
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     <td><ul>10 &micro;l</td>
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     <td>10 &micro;l</td>
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     <td><ul>1x</td>
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     <td>1x</td>
   </tr>
   </tr>

Revision as of 23:22, 4 October 2013

PCR

Materials:
  • PCR tubes
  • MQ water
  • DMSO 5%
  • Phusion buffer
  • DNTPs
  • F- and R-primer
  • Phusion Polymerase
  • DNA template


PCR reaction mixture:
Component 50 ml Final concentration
MQ water up to 50 µl
5x Phusion Buffer 10 µl 1x
    DMSO 5%
    1.5 µl
      10 mM dNTPs
      1 µl
      200 µM
      primer F
      2.5 µl
      0.5 µM
      primer R
      2.5 µl
      0.5 µM
      template DNA
      1 µl
      ∼1 ng
      Phusion Polymerase
      2 U/µl
      0.3 µl
      0.01 U/µl


    Cycling instructions:
      Temperature
      Time
      Number of Cycles
      98°C
      2 min
      1
      98°C
      10 sec
      34
      Tm-5°C
      25 sec
      72°C
      30 sec/kb
      72°C
      10 min
      1
      4°C
      ∞
      1

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