Team:Northwestern/PCR

Protocols

PCR

General Conditions:

  • 95°C (5 min) initial denaturation
  • ~25 cycles of:
    • 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
  • Running with Master Mix
    • 0.5 uL forward primer (10 uM)
    • 0.5 uL reverse primer (10 uM)
    • 1 uL DMSO
    • 1–5 ng template DNA (less works better)
    • 12.5 uL Master Mix
    • Nuclease-free water to 25 uL
  • Run 50 uL reactions to have more product