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RESEARCH DIVISION Laboratory Manual

 


 

Yeast Lysates for Westerns

  1. Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest. Spin cells down 2.6K for 5min.
  2. Resuspend in 1ml 0.25m NaOH/1% 2-mercaptoethanol
  3. Incubate 10min on ice.
  4. Add 0.16ml 50% trichloroacetic acid
  5. Incubate for 10min on ice.
  6. Pellet 14K 10min.
  7. Resuspend pellet in 1ml ice cold acetone.
  8. Pellet 14K 10min.
  9. Air dry pellet and resuspend in 200-500ul SDS SB and proceed as described below in section on western transfer.
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