Colony PCR from yeast, either to check inserts etc. or for sequencing.
- Lyticase (from Sigma)
- PCR buffers, primers, polymerase, etc.
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
- 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.
- 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.