Smolke:Protocols/Yeast Colony PCR

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Overview Colony PCR from yeast, either to check inserts etc. or for sequencing. Travis' Version (Lyticase lysis) 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. Dilute stock of lyticase to 50 U/mL in TE. Aliquot lyticase in 50uL quantities Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony Incubate at 37°C for 30 min Incubate at 95°C for 10 min Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction Notes I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards. The amount of yeast doesn't seem to matter much. I get success with very small colonies or with loads of cells. Josh's Version (NaOH lysis) Materials 20 mM NaOH PCR materials Procedure Aliquot 20uL NaOH into PCR tubes Pick colonies (I use pipet tips) into the NaOH Incubate at 95C for ~45 minutes Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge) Use 1uL of supernatant as template in a (10uL) PCR.