Team:Groningen/protocols/PCR

From 2013.igem.org

(Difference between revisions)
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<h5>PCR reaction mixture:</h5>
<h5>PCR reaction mixture:</h5>
<table border="1">
<table border="1">
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   <tr width="15%">
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   <tr>
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    <th >Component</th>
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    <th align="center "width="25%">Component</th>
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     <th >50 &micro;l</th>
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     <th align="center "width="25%">50 &micro;l</th>
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     <th >Final concentration</th>
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     <th align="center "width="25%">Final concentration</th>  
   </tr>
   </tr>
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   <tr align="center">
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   <tr>
     <td><ul>MQ water</td>
     <td><ul>MQ water</td>
     <td><ul>up to 50 &micro;l</td>
     <td><ul>up to 50 &micro;l</td>
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   <tr>
   <tr>
     <td><ul>10 mM dNTPs</td>
     <td><ul>10 mM dNTPs</td>
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     <td><ul>1&micro;l</td>
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     <td><ul>1 &micro;l</td>
     <td><ul>200 &micro;M</td>
     <td><ul>200 &micro;M</td>
   </tr>
   </tr>

Revision as of 18:27, 12 September 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 µl 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