Team:Groningen/protocols/PCR

From 2013.igem.org

(Difference between revisions)
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</ul>
</ul>
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<h3>Steps:</h3>
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<h3>PCR reaction mixture</h3>
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<table border = "1">
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<table border="1">
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<tr><td>Component</td>
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  <tr>
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<td>50&#956;l reaction</td>
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    <th>Component</th>
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<td>Final concentration</td>
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    <th>50&micro;l</th>
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</tr>
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    <th>Final concentration</th>
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<tr><td>MQ water</td>
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  </tr>
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<td>up to 50&#956;l</td>
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-
<td></td>
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  <tr>
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</tr>
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    <td>MQ water</td>
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<tr><td>5x Phusion Buffer</td>
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    <td ALIGN=CENTER>up to 50&micro;l</td>
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<td>10&#956;l</td>
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    <td></td>
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<td>1x</td>
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  </tr>
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</tr>
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<tr><td>10mM dNTPs</td>
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  <tr>
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<td>1&#956;l</td>
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    <td>5x Phusion Buffer</td>
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<td>200&#956;M</td>
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    <td ALIGN=CENTER>10&micro;l</td>
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</tr>
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    <td>1x</td>
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<tr><td>F-primer</td>
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  </tr>
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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>
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    <td>DMSO 5%</td>
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<tr><td>R-primer</td>
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    <td ALIGN=CENTER>1.5&micro;l</td>
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<td>2.5&#956;l</td>
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    <td></td>
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<td>0.2&#956;M</td>
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  </tr>
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</tr>
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<tr><td>Template DNA</td>
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  <tr>
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<td>1&#956;l</td>
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    <td>10mM dNTPs</td>
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<td>~1ng</td>
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    <td ALIGN=CENTER>1&micro;l</td>
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</tr>
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    <td>200&micro;M</td>
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<tr><td>Phusion Pol. 2U/&#956;l</td>
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  </tr>
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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>
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    <td>primer F</td>
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<tr><td>DMSO 5&#37;</td>
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    <td ALIGN=CENTER>2.5&micro;l</td>
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<td>1.5&#956;l</td>
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    <td>0.5&micro;M</td>
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<td></td>
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  </tr>
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</tr>
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</table>
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  <tr>
 +
    <td>primer R</td>
 +
    <td ALIGN=CENTER>2.5&micro;l</td>
 +
    <td>0.5&micro;M</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>template DNA</td>
 +
    <td ALIGN=CENTER>1&micro;l</td>
 +
    <td>&sim;1ng</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Phusion Polymerase <br>2U/&micro;l</td>
 +
    <td ALIGN=CENTER>0.3&micro;l</td>
 +
    <td>0.01U/&micro;l</td>
 +
  </tr>
 +
 
 +
</table>  
<h3>Cycling instructions:</h3>
<h3>Cycling instructions:</h3>
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<table border = "1">
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<table border="1">
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<tr><td>Temperature</td>
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  <tr>
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<td>Time</td>
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    <th>Temperature</th>
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<td>Number of cycles</td>
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    <th>Time</th>
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</tr>
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    <th>Number of Cycles</th>
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<tr><td>98&deg;C</td>
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  </tr>
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<td>2min</td>
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<td>1</td>
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  <tr>
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</tr>
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    <td>98&deg;C</td>
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<tr><td>98&deg;C</td>
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    <td>2min</td>
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<td>10sec</td>
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    <td>1td>
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<td rowspan="3"> 34 </td>
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  </tr>
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</tr>
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<tr><td>primer annealing temperature</td>
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  <tr>
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<td>25sec</td>
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    <td>98&deg;C</td>
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</tr>
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    <td>10sec</td>
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<tr><td>72&deg;C</td>
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    <td rowspan="3">34</td>
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<td>30sec/kb</td>
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  </tr>
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</tr>
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<tr><td>72&deg;C</td>
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  <tr>
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<td>10min</td>
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    <td>T<sub>m</sub>-5&deg;C</td>
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<td>1</td>
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    <td>25sec</td>
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</tr>
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  </tr>
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<tr><td>4&deg;C</td>
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<td>&infin;</td>
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  <tr>
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<td>1</td>
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    <td>72&deg;C</td>
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</tr>
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    <td>30sec/kb</td>
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</table>
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  </tr>
 +
 
 +
  <tr>
 +
    <td>72&deg;C</td>
 +
    <td>10min</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

Revision as of 20:00, 27 July 2013

PCR

Materials:

  • PCR tubes
  • F- and R-primer
  • DNTPs
  • Phusion buffer
  • MQ water
  • Phusion Polymerase
  • DNA template

PCR reaction mixture

Component 50µl Final concentration
MQ water up to 50µl
5x Phusion Buffer 10µl 1x
DMSO 5% 1.5µl
10mM dNTPs 1µl 200µM
primer F 2.5µl 0.5µM
primer R 2.5µl 0.5µM
template DNA 1µl ∼1ng
Phusion Polymerase
2U/µl		 0.3µl 0.01U/µl

Cycling instructions:

Temperature Time Number of Cycles
98°C 2min 1td>
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