Team:Groningen/protocols/PCR

From 2013.igem.org

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     <th>Component</th>
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     <th width="30%">Component</th>
     <th width="30%">50 &micro;l</th>
     <th width="30%">50 &micro;l</th>
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     <th>Final concentration</th>
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     <th width="30%">Final concentration</th>
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     <td WIDTH="25">MQ water</td>
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     <td><ul>MQ water</td>
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     <td VALIGN="top">up to 50 &micro;l</td>
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     <td><ul>up to 50 &micro;l</td>
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     <td></td>
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     <td><dd></td>
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     <td>5x Phusion Buffer</td>
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     <td><dd>5x Phusion Buffer</td>
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     <td STYLE="margin-left: 15px">10 &micro;l</td>
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     <td><dd>10 &micro;l</td>
     <td>1x</td>
     <td>1x</td>
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Revision as of 14:21, 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 1µl 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