Blackburn:YeastColonyPCR

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

• Qiagen Taq Polymerase Kit with Q-solution

• A small yeast colony

• 0.02M NaOH (3uL per reaction)

Procedure

Yeast Cell Lysis

1. Aliquot 3uL of 0.02M NaOH into PCR tubes.

2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.

• If the solution is cloudy, you''ve added enough cells.

3. [optional] Quick-freeze the cells in liquid nitrogen

• I normally skip this step. Works just fine.

4. 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

1. 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

2. Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).

3. Run the following PCR cycle:

1. 30 sec at 94C

2. 30 sec at 55C (or appropriate annealing temperature)

3. 1 min/kbp at 72C

4. 5 min at 94C

5. 30 cycles of:

6. 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.