Team:Groningen/protocols/Colony PCR

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<h1>PCR</h1>
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<h1>Colony PCR</h1>
<h5>Materials:</h5>
<h5>Materials:</h5>
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   <tr>
   <tr>
     <th>Component</th>
     <th>Component</th>
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     <th>50&micro;l</th>
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     <th>20&micro;l</th>
     <th>Final concentration</th>
     <th>Final concentration</th>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>5x Phusion Buffer</td>
     <td>5x Phusion Buffer</td>
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     <td ALIGN=CENTER>10&micro;l</td>
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     <td ALIGN=CENTER>2&micro;l</td>
     <td>1x</td>
     <td>1x</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>primer F</td>
     <td>primer F</td>
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     <td ALIGN=CENTER>2.5&micro;l</td>
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     <td ALIGN=CENTER>1&micro;l</td>
     <td>0.5&micro;M</td>
     <td>0.5&micro;M</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>primer R</td>
     <td>primer R</td>
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     <td ALIGN=CENTER>2.5&micro;l</td>
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     <td ALIGN=CENTER>1&micro;l</td>
     <td>0.5&micro;M</td>
     <td>0.5&micro;M</td>
   </tr>
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     <td>98&deg;C</td>
     <td>98&deg;C</td>
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     <td ALIGN=CENTER>2min</td>
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     <td ALIGN=CENTER>10min</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>

Revision as of 11:09, 28 July 2013

Colony PCR

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


PCR reaction mixture:
Component 20µl Final concentration
MQ water up to 50µl
5x Phusion Buffer 2µl 1x
DMSO 5% 1.5µl
10mM dNTPs 1µl 200µM
primer F 1µl 0.5µM
primer R 1µl 0.5µM
template DNA 1µl ∼1ng
Phusion Polymerase
2U/µl		 0.3µl 0.01U/µl


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

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