PCR Amplification of DNA

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Revision as of 16:57, 8 June 2020 by Reddj (Talk | contribs)

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SOP

Materials

Protocol

  • Use the following volumes per reaction
  • 12.5 uL DreamTaq
  • 7.5 uL RNAse-Free water
  • 5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))

PCR Program

  • 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)