"
USTC/Notebook/PCR&Colony PCR
From 2008.igem.org
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
