Yeast Colony PCR TopTaq

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Colony PCR of yeast cells using TopTaq

Because QIAGEN for some unknown reason (probably money) discontinued the TopTaq kits, we will finish the current kits and then move over to use a new, cheaper and easier kit from Nippon-Genetics. For that protocol see HERE.

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 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.

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 1 μl
MgCl2 (25mM) 0.2 μl
H2O 4.52 μl
Primer Fwd (10 μM) 0.4 μl
Primer Rev (10 μM) 0.4 μl
Q solution 5X 2 μl
CoralLoad 10X 1 μl
dNTPs (10 mM each) 0.4 μl
TopTaq polymerase (5U/μl) 0.08 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

This can be modified to 1kb / min