Yeast Colony PCR TopTaq

Colony PCR of yeast cells using TopTaq

This is an easy and quick protocol, but 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 and add some fresh yeast cells to the mix using pipette tips (so not toothpicks).
We often are left with a lot of buffers when we run out of the TopTaq enzym. Just use these buffers and replace the TopTaq for standard Invitrogen Taq; use 0.25μl per reaction.

Do sequencing reaction:
TopTaq PCR Buffer 10X	2.5 μl
MgCl2 (25mM)	0.5 μl
H2O	11.25 μl
Primer Fwd (10 μM)	1 μl
Primer Rev (10 μM)	1 μl
Q solution 5X	5 μl
CoralLoad 10X	2.5 μl
dNTPs (10 mM each)	1 μl
TopTaq polymerase (5U/μl)	0.2 μl

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

Number of cycles	Temperature	Duration
1 X	94°C	2 min
35X	94°C	30 sec
	52°C	30 sec
	72°C	2 min 30 sec
1X	72°C	7 min
	7°C	Inf