Freimoser:Protocols:FastYeastTransformation

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Revision as of 21:48, 9 May 2007

Protocol: Fast yeast transformation

1. Add 50 µl carrier DNA to a 1.5 ml tube.
2. scrap cells from plate and add to the carrier DNA.
3. Add in the following order:
   1. 1-2 µl DNA-plasmid (up to 30-50 µl)
   2. 240 µl PEG (50%)
   3. 36 µl Li-Ac (1M)
4. resuspend with blue tip
5. incubate at 30°C or RT at least 30 min
6. heat shock: 45°C, 15 min (or 42°C for 20 min)
7. spin down, resuspend in 100μl 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 3'350) solution, filter-sterilized
• 1M Li-Acetate, autoclaved

Reference

1. Gietz D, St Jean A, Woods RA, and Schiestl RH. . pmid:1561104. PubMed HubMed [Gietz-1992]