PCR Amplification with Platinum Taq High Fidelity

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Materials

  • Platinum PCR SuperMix High Fidelity (Life Technologies cat# 12532-016)
  • Amplification Primers (2 uM stock solution with both primers combined):

Amplification of Target Region

  • Using Platinum Taq High Fidelity amplify region of interest (design primers, should be <1000bp):
  • Per reaction:
    • 21.5 uL Platinum Taq High Fidelity Master Mix
    • 2.5 uL of Primers from a 2 uM stock solution
    • 1 uL of the Extracted DNA from the previous step.
  • The PCR program should be
    • 2 min at 95C
    • 94C for 15
    • 55C for 15s (adjust based on Tm of primers, use 5C less)
    • 68C for 1min/kb amplicon
    • Repeat steps 2-5 35X
    • 68C for 10min