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PCR Amplification of DNA

Revision as of 13:09, 5 May 2009 by Davebridges (Talk | contribs)

Revision as of 13:09, 5 May 2009 by Davebridges (Talk | contribs)

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
  • dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL)
  • Template – generally 1uL or less of a plasmid miniprep
  • Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

Protocol

  1. Use the following volumes per reaction
  • Buffer, 5 uL of 10X buffer
  • Primers, 10uL of 1uM stock solution in water (both primers combined)
  • dNTPs, 5uL of 2 mM
  • Sterile water, 28 uL
  • Template 1 uL
  • Polymerase 1 uL
  • Run PCR Program. 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)
Last modified on 8 June 2020, at 16:57