The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
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.
- 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* for 30 min
- Incubate at 95* 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.
Contact
Travis
|
