PCR Amplification of DNA

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Materials

  • Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
  • dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
  • Template – generally 1uL or less of a plasmid miniprep
  • Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

Protocol

  • Use the following volumes per reaction
  • Buffer, 5 uL of 10X buffer (Dave's fridge)
  • Primers, 10uL of 1uM stock solution in water (both primers combined)
  • dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
  • Sterile water, 28 uL
  • Template 1 uL
  • Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)
  • Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR (DAVETD) as follows:
  1. 1 min at 94
  2. 30s at 65
  3. 2 min/kb at 72
  4. 30s at 94
  5. 30s at 63 then -0.5/cycle
  6. 2 min/kb at 72
  7. Repeat steps 4-6 28 times
  8. 30s at 94
  9. 30s at 45
  10. 11 min at 72
  11. Hold at 4 until ready
  • Purify PCR product if necessary using Qiagen kit (Add 5x PB)