Team:Groningen/protocols/PCR

From 2013.igem.org

(Difference between revisions)
Line 72: Line 72:
<h3>Cycling instructions:</h3>
<h3>Cycling instructions:</h3>
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+
<table border = "1">
 +
<tr><td>Temperature</td>
 +
<td>Time</td>
 +
<td>Number of cycles</td>
 +
</tr>
 +
<tr><td>98&deg;C</td>
 +
<td>30sec</td>
 +
<td>1</td>
 +
</tr>
 +
<tr><td>98&deg;C</td>
 +
<td>10sec</td>
 +
<td>30</td>
 +
</tr>
 +
<tr><td>primer annealing temperature</td>
 +
<td>30sec</td>
 +
<td>30</td>
 +
</tr>
 +
<tr><td>72&deg;C</td>
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<td>30sec/kb</td>
 +
<td>30</td>
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</tr>
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<tr><td>72&deg;C</td>
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<td>10min</td>
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<td></td>
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</tr>
 +
<tr><td>4&deg;C</td>
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<td>&infin;</td>
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<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 09:22, 26 July 2013

PCR

Materials:

  • PCR tubes
  • F- and R-primer
  • DNTPs
  • Phusion buffer
  • H2O
  • Phusion Polymerase
  • DNA template

Steps:

Component 50μl reaction Final concentration
H2O up to 50μl
5x Phusion Buffer 10 1x
10mM dNTPs 1 200μM
F-primer 1 0.2μM
R-primer 1 0.2μM
Template DNA 1μl ~1ng
Phusion Pol. 0.5μl 0.02U/μl

Cycling instructions:

Temperature Time Number of cycles
98°C 30sec 1
98°C 10sec 30
primer annealing temperature 30sec 30
72°C 30sec/kb 30
72°C 10min
4°C ∞ 1

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