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RESEARCH DIVISION Laboratory Manual

 
 

Transformation of Yeast

  1. Pick a single yeast colony from the plate and incubate in 10 ml YEPD at 30° C overnight with shaking.
  2. Add 1 ml of the overnight culture into 50 ml YEPD and incubate at 30° C overnight, ODª1.5.
  3. Transfer about 30 - 40 ml of this culture to 300 ml YEPD to produce an OD=0.2. Incubate at 30° C with shaking for 3 - 4 hours.
  4. Pellet the cells in a GSA rotor at 5000 rpm for 5 min.
  5. Discard the supernatant and resuspend the pellet in 10 ml H2O. Transfer the cells to a 50 ml centrifuge tube.
  6. Pellet the cells in a SS34 rotor at 7000 rpm for 5 min.
  7. Discard the supernatant and resuspend the pellet in 30 ml sterile 1 X TE/LiAC (made fresh from stocks of 10X TE (0.1 M Tris-HCl, 0.01 M EDTA, pH7.5) and 10X LiAC (1M Lithium acetate adjusted to pH7.5 with dilute acetic acid, Sigma #L6883), and incubate at 30°C for 15 - 20 min with shaking.
  8. Pellet the cells in a SS34 rotor at 7000 rpm for 5 min.
  9. Discard the supernatant and resuspend the pellet in 1.5 ml sterile 1X TE/LiAC.
  10. Add the plasmid DNAs (5-10 g) and single-stranded carrier DNA (20ug) to a microfuge tube. The carrier DNA is produced by dissolving salmon sperm DNA in TE, sonicating it to reduce its viscosity, extracting it with phenol/chloroform, and precipitating it with ethanol. The DNA is resuspended at a concentration of 5 mg (or 10 mg)/ml in TE, placed in a boiling water bath for 20 min, and immediately cooled on ice. The carrier DNA solution can be stored in aliquots at -20°C.
  11. Add 100ul of the yeast suspension to each microfuge tube.
  12. Add 1.2 ml PEG solution (40% PEG, 1X TE, 1X LiAC, made fresh from stocks of 50% PEG 4000, Sigma P3640 3: 350; 10X TE, 10X LiAC) to each tube and incubate at 30°C for 30 min with sharking.
  13. Add dimethyl sulfoxide to 10%. Mix gently.
  14. Heat in a 42°C waterbath for 15 min.
  15. Pellet the cells at 7000 rpm for 2 min, aspirate the supernatant, resuspend the cells in 1 ml YEPD, and incubate at 30°C for 1 hour with shaking.
  16. Pellet the cells at 7000 rpm for 5 min and wash once with 1 ml TE.
  17. Pellet the cells and resuspend in 0.5 ml TE. Plate 0.1 ml onto each plate (0.5 ml for screening the cDNA library).
  18. Incubate the plates at 30°C until colonies appear.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998