Team:EPF-Lausanne/Protocol/PCR

From 2012.igem.org

(Difference between revisions)
Line 7: Line 7:
<!-- Don't forget to add this protocol to the protocol list page: -->
<!-- Don't forget to add this protocol to the protocol list page: -->
<!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol              -->
<!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol              -->
 +
1X Mastermix 20μl reaction
-
* Get the following excel: [[File:Team-EPF-Lausanne_Protocol_PCR.xls]]
+
# Water 20μl - x
-
* Fill in the pink fields with the appropriate values.
+
# HF-Buffer (5x) 4μl
-
** The annealing temperature is generally the lowest Tm. For long primers, use lowest Tm-5
+
# DMSO (optional) 0.6μl
-
** The extension time is 15-30s per 1kb
+
#dNTPs 0.4μl
-
* Prepare the master mix by adding the elements in the following order:
+
#Primers (50μM)
-
** H20
+
## Forward 0.2μl
-
** 5x HF Buffer
+
## Reverse 0.2 μl
-
** dNTP
+
#Template (10ng/μl) 0.5μl
-
** Polymerase (keep it cold!)
+
#Phusion HF polymerase 0.2μl
-
* Put 50ul of the master mix in a PCR tube for each different PCR
+
-
* Add the primers and the DNA
+
-
* Start the PCR!
+
{{:Team:EPF-Lausanne/Template/ProtocolFooter}}
{{:Team:EPF-Lausanne/Template/ProtocolFooter}}
<noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude>
<noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude>

Revision as of 09:52, 13 September 2012

Protocol: PCR

1X Mastermix 20μl reaction

  1. Water 20μl - x
  2. HF-Buffer (5x) 4μl
  3. DMSO (optional) 0.6μl
  4. dNTPs 0.4μl
  5. Primers (50μM)
    1. Forward 0.2μl
    2. Reverse 0.2 μl
  6. Template (10ng/μl) 0.5μl
  7. Phusion HF polymerase 0.2μl


Wiki Links

Important pages

Pages

Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Protocol/PCR"