PCR Amplification with Platinum Taq High Fidelity

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