Team:Northwestern/PCR

Protocols

PCR

General Conditions:

  • 95°C (5 min) initial denaturation
  • ~25 cycles of:
    • 95°C (7 s) denaturing
    • 50—70°C (7–15 s) annealing
      • Annealing temperature depends on the primers being used
    • 72°C (30—60 s per kb) elongation
      • Elongation time depends on the length of the sequence being amplified
  • 72°C (5 min) final incubation
  • * Touchdown PCR: Start 10°C above annealing temperature and decrease the Ta over 10 cycles to the final primer Ta if primers are binding to alternate sites and giving incorrect products

Recipe (25 uL reaction):

  • 2.5 µL 10x buffer
  • 0.5 µL 10mM dNTPs
  • 0.5 µL forward primer (10 uM)
  • 0.5 uL reverse primer (10 uM)
  • 1–5 ng template DNA (less works better)
  • 1 uL DMSO
  • 0.25 µL polymerase
  • Nuclease-free water to 25 uL

Using Master Mix (25 uL reaction):

  • 0.5 uL forward primer (10 uM)
  • 0.5 uL reverse primer (10 uM)
  • 1–5 ng template DNA (less works better)
  • 1 uL DMSO
  • 12.5 uL Master Mix
  • Nuclease-free water to 25 uL