Phusion colony PCR

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 colony PCR see here.

Prepare mastermix:

Do sequencing reaction:
Phusion HF Buffer 5X	4 μl
H2O	9 μl
dNTPs (10 mM each)	0.8 μl
Primer Fwd (10 μM)	0.8 μl
Primer Rev (10 μM)	0.8 μl

Divide over PCR reaction tubes/wells (15.5μl per sample) 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
• 4 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 (can be changed to 2kb/min)
1X	72°C	7 min
	7°C	Inf