Smolke:Protocols/Yeast Colony PCR

Overview
Colony PCR from yeast, either to check inserts etc. or for sequencing.

Materials

 * Lyticase (from Sigma)
 * TE
 * PCR buffers, primers, polymerase, etc.

Procedure
The basic idea is breaking the cells with lyticase and heat, then doing PCR.


 * 1) Dilute stock of lyticase to 50 U/mL in TE.
 * 2) Aliquot lyticase in 50uL quantities
 * 3) Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
 * 4) Incubate at 37&deg;C for 30 min
 * 5) Incubate at 95&deg;C for 10 min
 * 6) Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction

Materials

 * 20 mM NaOH
 * PCR materials

Procedure

 * 1) Aliquot 20uL NaOH into PCR tubes
 * 2) Pick colonies (I use pipet tips) into the NaOH
 * 3) Incubate at 95C for ~45 minutes
 * 4) Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge)
 * 5) Use 1uL of supernatant as template in a (10uL) PCR.