FCCT Biochemistry Lab:PCR protocol iGEM HS

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Protocols

PCR

Amplification PCR with Taq polymerase

Setup Amplification PCR Reaction

  • 1 μL Template DNA
  • 11 μl deionized (dI) H2O
  • 1 μl 10μM forward primer
  • 1 μl 10μM reverse Primer
  • 2 μl 10x Taq Polymerase Buffer
  • 2 μl MgCl2
  • 1 μl 10x dilluted taq polymerase

Total 20μl

Amplification PCR Reaction Program

  • 1. 95 °C for 5min
  • 2. 95 °C for 30s
  • 3. 50 °C for 90s (important step)
  • 4. 72 °C for 1min per kb target gene plus 10-15% (important step)
  • 5. Repeat 2-4 25x
  • 6. 72 °C for 5 to 10min
  • 7. 4 °C for forever (don’t forget to turn the machine off the next day)