Two Hybrid Screening Problem

Thomas O'Neill toneil1 at gl.umbc.edu
Fri Apr 7 15:10:40 EST 1995


On Fri, 7 Apr 1995 wvcrieki at lmb1.rug.ac.be wrote:

> Hello ...,
> 
> In using CG-1945 for a two hybrid screening I'm having trouble in obtaining
> colonies big enough to lift (for the lysis in liquid nitrogen). The colonies 
> start growing after 2-3 days but stop after 5 days. How can I overcome this ? 
> e.g. changing X-gal assay, or using different plating techniques (I'm using SD 
> plates lacking LEU,TRP & HIS, 5 mM 3-TA). All suggestions are welcome.
> 
> Thanks for your time
> Wim Van Criekinge


  Rather than doing an X-gal filter assay at this step, I prepare 
phoshate- buffered plates, and spread X-gal directly on the plate before 
plating the yeast.

  Prepare a 1M solution of KH2PO4 pH 7.0 and add to molten agar to a 
final conc. of 100 mM.  This buffering allows the Bgal enzyme to function, 
however the yeast cells do not grow quite as well at neutral pH. Less 
Xgal seems to work better than more. Try 25 ul of a 2% stock per 100mm plate.

				Good luck,
				Thomas J. O'Neill




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