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RESEARCH DIVISION Laboratory Manual

 


 

Modified Yeast Transformation

  1. Inoculate cells from an overnight culture into 50 ml YEPD and incubate at 30C with shaking. Typically, add 0.1 to 0.2 ml saturated culture in the evening to get an of OD600 = 1 to 2 the next morning.
  2. Transfer enough of this culture to 300 ml YEPD to produce an OD6oo=0.2 (ca. 20 mls.). Incubate at 30C to an OD600 of 6.5 to 8.5. I often grow three cultures overnight starting with different amounts of inoculum and use the one at the best OD in the morning.
  3. Pellet the cells in a GSA rotor (5000 rpm, 5 min., RT).
  4. Discard the supernatant and resuspend the pellet in 10 ml T.E. Transfer the cells to a 50 ml centrifuge tube.
  5. Pellet the cells in an SS34 rotor (7000 rpm, 5 min., RT).
  6. Discard the supernatant and resuspend the pellet in 10 ml sterile 1 X TE/LiAC (made fresh from stocks of 10 X TE [0.1 M Tris-HCI, 0.01 M EDTA, pH 7.5] and 10X LiAC [1 M lithium acetate adjusted to pH 7.5 with dilute acetic acid]. Repeat.
  7. Pellet cells in microfuge at 7000rpm, discard sup. and resuspend in 1.5mL TE/LiAC.
  8. Add 100ul cells to the plasmid DNAs (1-5 ug) and single-stranded carrier DNA (20ug) in 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 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-20C.
  9. Add 700ul ml sterile PEG solution (40 % PEG, 1 X TE, 1 XLiAC, made fresh from stocks of 50 % PEG 4000, 10 X TE, 10 X LiAC) to each tube and incubate at 30C for 45min. up to 1.5hrs (longer is fine if maximum efficiency is not required; 1.5hrs is optimum) with shaking.
  10. Add dimethyl sulfoxide to 10% (70uL). Mix gently.
  11. Heat pulse for 15 min. in a 42C waterbath.
  12. Pellet cells in microfuge at 7000rpm, pour off most of sup and plate on appropriate medium.
  13. Incubate the plates at 30C until colonies appear, usually 2-3 days.
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