Smolke:Protocols/Yeast Colony PCR

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.

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°C for 30 min
5. Incubate at 95°C for 10 min
6. 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

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.