Freimoser:Protocols:FastYeastTransformation

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Revision as of 08:46, 17 October 2006

Protocol: Fast yeast transformation

1. 50 µl carrier DNA
2. scrap cells from plate
3. 1-2 µl DNA-plasmid (up to 30-50 ul)
4. 240 µl PEG (50%)
5. 36 µl Li-Ac (1M)
6. resuspend with blue tip
7. incubate at 30°C or RT at least 30 min
8. heat shock: 45°C, 15 min (or 42°C for 20 min)
9. spin down, resuspend in 100ul H2O and plate everything on corresponding medium

Material:

• Plasmid: 100 ng - 1 µg
• carrier DNA: salmon/herring sperm DNA in H2O, 2mg/ml stock, 500 µl aliquots, heat-inactivated at 95°C for 5-6 min and put on ice before use (has to be done once).
• 50% poly-ethylen-glycol (PEG) (MW 33-50) solution, filter-sterilized
• 1M Li-Acetate, autoclaved

Reference

1. . Acid Res.-1992 pmid=1561104 [Gietz-Nuc]