Team:Groningen/protocols/Colony PCR

From 2013.igem.org

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<br>
<br>
<h5>PCR reaction mixture:</h5>
<h5>PCR reaction mixture:</h5>
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<table id="normal">
+
<table id="normal" width="60%">
   <tr>
   <tr>
     <th>Component</th>
     <th>Component</th>
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   <tr>
   <tr>
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     <td width="500px">MQ water</td>
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     <td>MQ water</td>
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     <td ALIGN=CENTER>up to 20 &micro;l</td>
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     <td>up to 20 &micro;l</td>
     <td></td>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>10 mM dNTPs</td>
     <td>10 mM dNTPs</td>
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     <td ALIGN=CENTER>1 &micro;l</td>
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     <td>1 &micro;l</td>
     <td>200 &micro;M</td>
     <td>200 &micro;M</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>primer F</td>
     <td>primer F</td>
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     <td ALIGN=CENTER>1 &micro;l</td>
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     <td>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>1 &micro;l</td>
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     <td>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>template DNA</td>
     <td>template DNA</td>
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     <td ALIGN=CENTER>1 &micro;l</td>
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     <td>1 &micro;l</td>
     <td>&sim;1 ng</td>
     <td>&sim;1 ng</td>
   </tr>
   </tr>
   <tr>
   <tr>
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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>
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     <td ALIGN=CENTER>0.3 &micro;l</td>
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     <td>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>
   </tr>
   </tr>
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<br>
<br>
<h5>Cycling instructions:</h5>
<h5>Cycling instructions:</h5>
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<table id="normal">
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<table id="normal" width="60%">
   <tr>
   <tr>
     <th>Temperature</th>
     <th>Temperature</th>
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   <tr>
   <tr>
     <td>98&deg;C</td>
     <td>98&deg;C</td>
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     <td ALIGN=CENTER>10 min</td>
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     <td>10 min</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>98&deg;C</td>
     <td>98&deg;C</td>
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     <td ALIGN=CENTER>10 sec</td>
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     <td>10 sec</td>
     <td rowspan="3">34</td>
     <td rowspan="3">34</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>T<sub>m</sub>-5&deg;C</td>
     <td>T<sub>m</sub>-5&deg;C</td>
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     <td ALIGN=CENTER>25 sec</td>
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     <td>25 sec</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <td>72&deg;C</td>
     <td>72&deg;C</td>
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     <td ALIGN=CENTER>30 sec/kb</td>
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     <td>30 sec/kb</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <td>72&deg;C</td>
     <td>72&deg;C</td>
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     <td ALIGN=CENTER>10 min</td>
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     <td>10 min</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>4&deg;C</td>
     <td>4&deg;C</td>
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     <td ALIGN=CENTER>&infin;</td>
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     <td>&infin;</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>

Latest revision as of 23:27, 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