Blackburn:Yeast Colony PCR

Overview

This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.

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 sometime. 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. 5 min at 94C
   2. 30 cycles of:
      1. 30 sec at 94C
      2. 30 sec at 55C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C
   3. 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.

Contact

Tet (Blackburn Lab)