Yeast Colony PCR FastGene Nippon

This is an easy and quick protocol, similar to the TopTaq protocol, but better, cheaper and easier! Nevertheless, it does not always work for complex amplicons or amplicons over 2.5kb long, for which Phusion colony PCR works better. For E. coli see here.

Prepare mastermix, divide over PCR reaction tubes/wells (10µl per sample) and add some but not too many fresh yeast cells (the mix should become somewhat cloudy) to the mix using pipette tips (so not toothpicks).

We have optimized this to use only 10μl per reaction to reduce costs.

Aliquot! We noticed that FastGene is not stable for many Freeze Thaw cycles. When opening a new tube, Aliquot the whole mix out into Eppies, using 185µl per tube (enough for 32 reactions). Freeze these in middle drawer freezer 7.

Component	Per sample	Volumes MasterMix for 16 samples
H2O	4.2 μl	70.5 μl
FastGene	5 μl	84 μl
Primer Fwd (10 μM)	0.4 μl	6.7 μl
Primer Rev (10 μM)	0.4 μl	6.7 μl

Run using program (bart -> pombe -> colony PCR)

Number of cycles	Temperature	Duration
1 X	94°C	2 min
35X	94°C	30 sec
	57°C *	30 sec
	72°C	2 min 30 sec†
1X	72°C	7 min
	7°C	Inf

* This is a good starting temperature for most Tm = 60°C primers, but might need to be tweaked.

^†This can be modified to 1kb / min