Berkeley LBL/PCRextaq

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PCR (TaKaRa Ex Taq Polymerase)

1. Set Up PCR Reaction:

      1 uL template DNA
      5 uL Ex Taq Buffer
      4 uL  dNTP mixture (2.5mM each)
      5 uL Primer Mix
      0.5 uL DMSO (optional)
      0.25 uL TaKaRa Ex Taq Polymerase
      Add H2O to total volume 50 uL

2. Run PCR machine

  • Use 1 min/kb for extension
  • General cycle:
      1. Initial Denature	98ºC 			30 sec
      2. Denaturation	        98 ºC	   		8 sec	
      3. Annealing		Annealing Temp	30 sec	
      4. Extension		72 ºC			1 min/kb
      5. Repeat steps 2-4 for an additional 29 cycles
      6. Final Extension	72 ºC			10 min
      7. 			4 ºC 			forever
  • Immediately place on ice or in 4 ºC after removing from PCR machine
  • PCR reactions should be set up on ice.
  • Add the polymerase last and carefully.
  • DMSO is used only for high GC content