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RESEARCH DIVISION Laboratory Manual

 


 

Filter Assay for the Detection of Yeast Colonies Producing

Preparation of stock solutions

Z buffer (per litre)

Na2HPO4.7H2O
OR Na2HPO4
16.1 g
8.5 g
NaH2PO4.H2O 5.5 g
KCl 0.75 g
MgSO4.7H2O 0.246 g
Adjust pH to 7.0

X-gal

Dissolve 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) in N,N-dimenthylformamide at a concentration of 20 mg/ml.

Procedure

  1. Prepare a X-gal/Z buffer solution containing 100 ml of Z buffer, 0.27 ml of 2-mercaptoethanol, and 1.67 ml X-gal stock. To assay colonies on a 100 mm diameter dish, pipet 1.8 ml of this solution to a clean dish and soak a filter placed onto the solution. Use either Whatman 1 or VWR grade 413 paper filters.
  2. After the colonies have grown, they are picked and transferred to a filter (or place a sterile filter onto a transformation plate and orient it).
  3. The filter is placed into a pool of liquid nitrogen for 10 seconds, then removed and allowed to thaw at room temperature.
  4. Carefully layer the thawed filter, colony side up, onto the presoaked with X-gal/Z buffer solution. Colonies producing b-galactosidase will turn blue any time from 30 min to 30 hours.
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