Team:Groningen/protocols/PCR

From 2013.igem.org

(Difference between revisions)
Line 46: Line 46:
</tr>
</tr>
<tr><td>5x Phusion Buffer</td>
<tr><td>5x Phusion Buffer</td>
-
<td>10</td>
+
<td>10&#956;l</td>
<td>1x</td>
<td>1x</td>
</tr>
</tr>
<tr><td>10mM dNTPs</td>
<tr><td>10mM dNTPs</td>
-
<td>1</td>
+
<td>1&#956;l</td>
<td>200&#956;M</td>
<td>200&#956;M</td>
</tr>
</tr>
<tr><td>F-primer</td>
<tr><td>F-primer</td>
-
<td>1</td>
+
<td>1&#956;l</td>
<td>0.2&#956;M</td>
<td>0.2&#956;M</td>
</tr>
</tr>
<tr><td>R-primer</td>
<tr><td>R-primer</td>
-
<td>1</td>
+
<td>1&#956;l</td>
<td>0.2&#956;M</td>
<td>0.2&#956;M</td>
</tr>
</tr>

Revision as of 10:19, 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 10μl 1x
10mM dNTPs 1μl 200μM
F-primer 1μl 0.2μM
R-primer 1μl 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
72°C 30sec/kb
72°C 10min 1
4°C ∞ 1

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