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Team:BIOTEC Dresden/Protocols:Polymerase Chain Reaction (high fidelity)
From 2010.igem.org
(use this procedure only if your amplified DNA needs to be highly accurate (for downstream digestions, ligations, sequencing etc)
Reaction volume = 50μL, scale accordingly
Materials
- sterile 0.2mL PCR strips or plates
- ultra-pure water
- 5X HF Buffer
- 10mM dNTPs
- forward primers
- reverse primers
- template DNA
- Phusion Polymerase
Procedure
- 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).
