USTC/Notebook/PCR&Colony PCR

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'''[[Team:USTC/Notebook|< Back to Notebook]]'''
'''[[Team:USTC/Notebook|< Back to Notebook]]'''

Revision as of 14:03, 27 October 2008


< Back to Notebook

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

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