(Difference between revisions)
Revision as of 17:46, 24 March 2012
Adapted from Gietz, Nature Protocols 2007 (dx.doiorg/10.1038/nprot.2007.14), this works similarly to the standard, longer method. -Leo
- Sterile water
- 1M Lithium Acetate
- 50% PEG (4000)
- Salmon sperm DNA ('SSD', 10 mg/mL, stored at -20C)
- Plasmid DNA
- Grow an overnight culture of your chosen yeast strain in appropriate media.
- Alternatively, you can grow serial dilutions of your chosen strain, and one of them will likely be at the appropriate OD the next morning. For me, picking some cells into 3 mL of media, then taking 300 ul of this into a 25 mL flask works pretty well. -Leo
- Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice.
- Pellet cells at ~6000g for 1 minute. Discard supernatant
- Add to the pellet the following, in order, then quickly vortex. If doing multiple transformations, you can make a master mix of everything except the DNA and aliquot.
- 240 ul 50% PEG
- 36 ul 1M LiAc
- 10 ul single-stranded carrier DNA (10 mg/ml)
- 74 ul of sterile water plus any plasmid DNA
- Incubate in 42°C water bath 15-40 minutes
- Spin 30 seconds at max speed and carefully remove supernatant
- Resuspend in 100-200 ul sterile water by vortexing and plate on appropriate plates.
- Let grow at 30°C 2-3 days
Colonies should be ready. Some strains/plasmids may take an extra day.