USTC/Notebook/PCR&Colony PCR

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2. add the following components:
2. add the following components:
-
- 2µL  PCR buffer (rock gently after thawing, quick spin before use)
+
- 2µL  PCR buffer (rock gently after thawing, quick spin before use)
-
- 1.2uL  Mgcl2
+
- 1.2uL  Mgcl2
- 0.4uL  dNTPs
- 0.4uL  dNTPs
- 200nM final concentration of each primer  
- 200nM final concentration of each primer  

Revision as of 13:45, 27 October 2008

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PCR

Briefly, a typical reaction is set up as follows:


1. set up pre-labeled reaction tubes on ice

2. add the following components:

- 2µL  PCR buffer (rock gently after thawing, quick spin before use)
- 1.2uL  Mgcl2

- 0.4uL dNTPs - 200nM final concentration of each primer - 0.2uL Taq enzyme - template DNA (note: the total volume of PCR is 20µL)

3. make sure reaction tubes are properly capped before placing in thermocycler

4. perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C


colony PCR

- 0.2 uL primer #1 (to 25uM) - 0.2 uL primer #2 - 0.4 uL dNTPs - 2 uL Buffer - 5 uL colony culture - 0.2 uL Taq - 10.4 uL H20

perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min