Blackburn:Yeast Colony PCR

Updated Protocol: Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0

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)

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.
 * 3) *If the solution is cloudy, you've added enough cells.
 * 4) [optional] Quick-freeze the cells in liquid nitrogen
 * 5) *I normally skip this step. Works just fine.
 * 6) Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
 * 7) *In the mean time, prepare the master mix for the PCR reaction.
 * 8) *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:
 * 2) *5uL 5X Q-solution
 * 3) *2.5uL 10X PCR Buffer
 * 4) *0.5uL dNTPs (10mM each)
 * 5) *0.5uL foward primer (100uM)
 * 6) *0.5uL reverse primer (100uM)
 * 7) *0.25uL Taq
 * 8) *12.75uL ddH2O
 * 9) Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
 * 10) Run the following PCR cycle:
 * 11) 5 min at 94C
 * 12) 30 cycles of:
 * 13) 30 sec at 94C
 * 14) 30 sec at 55C (or appropriate annealing temperature)
 * 15) 1 min/kbp at 72C
 * 16) 10 min at 72C

Contact
Tet (Blackburn Lab)