Phusion colony PCR

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Colony PCR of yeast cells using Phusion

This is a slightly slower and more expensive protocol than the TopTaq protocol, but also more robust for longer or more complex amplicons. For E. coli see here.
Prepare mastermix:

Do sequencing reaction:
Phusion HF Buffer 5X 5 μl
H2O 12 μl
dNTPs (10 mM each) 1 μl
Primer Fwd (10 μM) 1 μl
Primer Rev (10 μM) 1 μl

Divide over PCR reaction tubes/wells and add some fresh yeast cells to the mix using pipette tips (so not toothpicks).

Boil the sample in the PCR machine at 98°C for 10 minutes and cool down to 7°C. Now add to each tube 5μl of a mix of:

  • 0.2 μl Phusion polymerase
  • 5 μl H2O

We dilute to Phusion to make the pipetting a bit easier and obtain the right final concentration for all ingredients.
Run using program (bart -> pombe -> colony PCR)

Number of cycles Temperature Duration
1 X 94°C 2 min
35X 94°C 30 sec
55-60°C 30 sec
72°C 2 min 30 sec
1X 72°C 7 min
7°C Inf