Team:Groningen/protocols/Colony PCR

From 2013.igem.org

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<h5>PCR reaction mixture:</h5>
<h5>PCR reaction mixture:</h5>
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     <th><ul>Component</th>
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     <td>Phusion Polymerase <br>2U/&micro;l</td>
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     <td>Phusion Polymerase <br>2 U/&micro;l</td>
     <td ALIGN=CENTER>0.3 &micro;l</td>
     <td ALIGN=CENTER>0.3 &micro;l</td>
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     <td>0.01U/&micro;l</td>
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     <td>0.01 U/&micro;l</td>
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<h5>Cycling instructions:</h5>
<h5>Cycling instructions:</h5>
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     <th>Temperature</th>
     <th>Temperature</th>

Revision as of 23:19, 4 October 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
    20 µl
    Final concentration
MQ water up to 20 µl
5x Phusion Buffer 2 µl 1x
DMSO 5% 1.5 µl
10 mM dNTPs 1 µl 200 µM
primer F 1 µl 0.5 µM
primer R 1 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 10 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