Team:Groningen/protocols/PCR

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<div class="mainContent">
<div class="mainContent">
-
<h1>PCR</h1>
+
<h2>PCR</h2>
-
<h3>Materials:</h3>
+
<h5>Materials:</h5>
<ul type="square">
<ul type="square">
<li>PCR tubes</li>
<li>PCR tubes</li>
-
<li>F- and R-primer</li>
 
-
<li>DNTPs</li>
 
-
<li>Phusion buffer</li>
 
<li>MQ water</li>
<li>MQ water</li>
 +
<li>DMSO 5%</li>
 +
<li>Phusion buffer</li>
 +
<li>DNTPs</li>
 +
<li>F- and R-primer</li>
<li>Phusion Polymerase</li>
<li>Phusion Polymerase</li>
<li>DNA template</li>
<li>DNA template</li>
</ul>
</ul>
-
<h3>Steps:</h3>
 
-
<table border = "1">
 
-
<tr><td>Component</td>
 
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<td>50&#956;l reaction</td>
 
-
<td>Final concentration</td>
 
-
</tr>
 
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<tr><td>MQ water</td>
 
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<td>up to 50&#956;l</td>
 
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<td></td>
 
-
</tr>
 
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<tr><td>5x Phusion Buffer</td>
 
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<td>10&#956;l</td>
 
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<td>1x</td>
 
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</tr>
 
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<tr><td>10mM dNTPs</td>
 
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<td>1&#956;l</td>
 
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<td>200&#956;M</td>
 
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</tr>
 
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<tr><td>F-primer</td>
 
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<td>2.5&#956;l</td>
 
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<td>0.2&#956;M</td>
 
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</tr>
 
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<tr><td>R-primer</td>
 
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<td>2.5&#956;l</td>
 
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<td>0.2&#956;M</td>
 
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</tr>
 
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<tr><td>Template DNA</td>
 
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<td>1&#956;l</td>
 
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<td>~1ng</td>
 
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</tr>
 
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<tr><td>Phusion Pol. 2U/&#956;l</td>
 
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<td>0.3&#956;l</td>
 
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<td>0.02U/&#956;l</td>
 
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</tr>
 
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<tr><td>DMSO 5&#37;</td>
 
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<td>1.5&#956;l</td>
 
-
<td></td>
 
-
</tr>
 
-
</table>
 
-
<h3>Cycling instructions:</h3>
+
 
-
<table border = "1">
+
<p>
-
<tr><td>Temperature</td>
+
<br>
-
<td>Time</td>
+
<h5>PCR reaction mixture:</h5>
-
<td>Number of cycles</td>
+
<table id="normal" width="60%">
-
</tr>
+
  <tr>
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<tr><td>98&deg;C</td>
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    <th>Component</th>
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<td>2min</td>
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    <th>50 ml</th>
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<td>1</td>
+
    <th>Final concentration</th>
-
</tr>
+
  </tr>
-
<tr><td>98&deg;C</td>
+
 
-
<td>10sec</td>
+
  <tr>
-
<td rowspan="3"> 34 </td>
+
    <td>MQ water</td>
-
</tr>
+
    <td>up to 50 &micro;l</td>
-
<tr><td>primer annealing temperature</td>
+
    <td></td>
-
<td>25sec</td>
+
  </tr>
-
</tr>
+
 
-
<tr><td>72&deg;C</td>
+
  <tr>
-
<td>30sec/kb</td>
+
    <td>5x Phusion Buffer</td>
-
</tr>
+
    <td>10 &micro;l</td>
-
<tr><td>72&deg;C</td>
+
    <td>1x</td>
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<td>10min</td>
+
  </tr>
-
<td>1</td>
+
 
-
</tr>
+
  <tr>
-
<tr><td>4&deg;C</td>
+
    <td>DMSO 5%</td>
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<td>&infin;</td>
+
    <td>1.5 &micro;l</td>
-
<td>1</td>
+
    <td></td>
-
</tr>
+
  </tr>
-
</table>
+
 
 +
  <tr>
 +
    <td>10 mM dNTPs</td>
 +
    <td>1 &micro;l</td>
 +
    <td>200 &micro;M</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>primer F</td>
 +
    <td>2.5 &micro;l</td>
 +
    <td>0.5 &micro;M</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>primer R</td>
 +
    <td>2.5 &micro;l</td>
 +
    <td>0.5 &micro;M</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>template DNA</td>
 +
    <td>1 &micro;l</td>
 +
    <td>&sim;1 ng</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Phusion Polymerase <br>2 U/&micro;l</td>
 +
    <td>0.3 &micro;l</td>
 +
    <td>0.01 U/&micro;l</td>
 +
  </tr>
 +
 
 +
</table>
 +
 
 +
<p>
 +
<br>
 +
<h5>Cycling instructions:</h5>
 +
<table id="normal" width="60%">
 +
  <tr>
 +
    <th>Temperature</th>
 +
    <th>Time</th>
 +
    <th>Number of Cycles</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>98&deg;C</td>
 +
    <td>2 min</td>
 +
    <td>1</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>98&deg;C</td>
 +
    <td>10 sec</td>
 +
    <td rowspan="3">34</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>T<sub>m</sub>-5&deg;C</td>
 +
    <td>25 sec</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>72&deg;C</td>
 +
    <td>30 sec/kb</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>72&deg;C</td>
 +
    <td>10 min</td>
 +
    <td>1</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>4&deg;C</td>
 +
    <td>&infin;</td>
 +
    <td>1</td>
 +
  </tr>
 +
</table>  
<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf
<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf

Latest revision as of 23:25, 4 October 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 ml 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