Freimoser:Protocols:YeastTransformation

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General comments

This protocol is used to clone DNA fragements and PCR products into the yeast genome by homologous recombination. It is a much simplified version of the high efficiency yeast transformation protocol. This protocol works usually fine to replace selection markers, create knockouts or add tags via homologous recombination. To transform yeast with plasmids we use the fast yeast transformation protocol.


Preparation of "competent" yeast cells

  1. Make a 3 mL overnight culture of the required strains in YPD
  2. The next day inoculate 5 mL YPD medium with 1 OD600 yeast cells (so that the final OD600 will be 0.2)
  3. Let this culture grow for 2-3 h (the OD600 should be 0.8-1.2, often we take the culture after 3 h without measuring the OD600 since this proofed fine for most strains)
  4. spin down the cells and resuspend the pellet in 5 mL H2O
  5. spin down the cells and resuspend the pellet in 1 mL 100 mM LiAc
  6. spin down the cells


Transformation

  1. Add the following to the pellet (in the order indicated):
    1. 240 μL PEG
    2. 36 μL LiAc (1M)
    3. 50 μL carrier DNA
    4. x μL DNA
    5. 34-x μL H2O
  2. vortex 1 min (if the pellet is not resuspended use a pipette)
  3. heat shock: 42°C, 40 min
  4. spin: 6000-8000 rpm, 15s
  5. remove liquid, add 800 μl YPD and leave 2 h at 30°C
  6. spin down cells (5000 rpm, 3 min) plate everything


Material:

  • carrier DNA: salmon/herring sperm DNA in H2O, 2 mg/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
  • 1 M Li-Acetate, autoclaved
  • 0.1 M Li-Acetate, autoclaved


Reference

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

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