Blackburn:Yeast Colony PCR
Overview
This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0
Materials
Standard PCR machine, tubes
A small yeast colony
0.02M NaOH (3uL per reaction)
Procedure
Yeast Cell Lysis
Aliquot 3uL of 0.02M NaOH into PCR tubes.
Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
If the solution is cloudy, you''ve added enough cells.
[optional] Quick-freeze the cells in liquid nitrogen
I normally skip this step. Works just fine.
Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
In the mean time, prepare the master mix for the PCR reaction.
The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
PCR
Prepare the master mix solution containing:
5uL 5X Q-solution
2.5uL 10X PCR Buffer
0.5uL dNTPs (10mM each)
0.5uL foward primer (100uM)
0.5uL reverse primer (100uM)
0.25uL Taq
12.75uL ddH2O
Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
Run the following PCR cycle:
30 sec at 94C
30 sec at 55C (or appropriate annealing temperature)
1 min/kbp at 72C
5 min at 94C
30 cycles of:
10 min at 72C
Notes
Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols.
The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
The expected PCR product should be as short as possible. Anything less than 1kbp can be easily amplified.
