Team:Brown-Stanford/Lab/Protocols/PhusionPCR

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Phusion® PCR

(Taken from the [http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Phusion® High-Fidelity DNA Polymerase Protocol]; this protocol was edited for amplification of [http://partsregistry.org/Part:BBa_K656013 Part BBa_K656013])

Reagents for 20 µL reactions

  • 11.8µL Nuclease-Free Water
  • 4µL 5x Phusion® HF Buffer
  • .4 µL 10 mM dNTP
  • 1 µL primer A (.5µM concentration recommended)
  • 1 µL primer B (.5µM concentration recommended)
  • 1 µL template DNA
  • .6µL DMSO (optional; recommended for GC-rich amplicons)
  • .2µL Phusion® DNA Polymerase


Thermocycle

  1. 98˚, 30 seconds - initial denaturization
  2. 98˚, 10 seconds - denaturation
  3. 72˚, 4 minutes - annealing and extension
  4. Repeat from Step 2 32x
  5. 72˚, 10 minutes - final extrension/cleanup
  6. 4˚, hold indefinitely
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