Team:BIOTEC Dresden/Protocols:Polymerase Chain Reaction (high fidelity)


(use this procedure only if your amplified DNA needs to be highly accurate (for downstream digestions, ligations, sequencing etc)

Reaction volume = 50μL, scale accordingly


  • sterile 0.2mL PCR strips or plates
  • ultra-pure water
  • 5X HF Buffer
  • 10mM dNTPs
  • forward primers
  • reverse primers
  • template DNA
  • Phusion Polymerase


  • Everything on ice!
  • Prepare a Master Mix:
  • into a 1.5mL microfuge tube, add the following per 50 μL reaction:
  • 38μL ultra pure water
  • 10μL 5X HF Buffer
  • 1μL 10mM dNTP
  • 0.25μL forward primer
  • 0.25μL reverse primer
  • 0.5 μL Phusion
  • Pipet 1μL (usually) of your DNA template into a sterile 0.2mL PCR strip or plate
  • Note: also prepare
  • one or two extra reactions to allow for pipetting errors.
  • Vortex Mastermix and pulse spin down
  • Pipet 50μL of your Mastermix into PCR tube
  • Pulse spin PCR reactions using the PCR strip centrifuge (in Anni’s lab; front bench)
  • Set up PCR machine according to need (denaturation temperature, elongation time).
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