USTC/Notebook/PCR&Colony PCR

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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: (note: add ddH2O to 20µL, the total volume of PCR is 20µL)
 * 2µL   PCR buffer (rock gently after thawing, quick spin before use)
 * 1.2uL MgCl2
 * 0.4uL dNTPs
 * 200nM final concentration of each primer VF2 and VR (0.2uL)
 * 0.2uL Taq enzyme
 * template DNA

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
 * 0.4 uL MgCl2
 * 2 uL PCR Buffer
 * 2 uL colony culture
 * 0.2 uL Taq enzyme
 * 15.8 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