Freimoser:Protocols:FastYeastTransformation

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