background in 2 hybrid system

Michael Moser moser at U.WASHINGTON.EDU
Thu Oct 26 11:36:29 EST 1995


Dear Ingrid

	Plenty of people have done a 2-H screen using only lacZ as an
indicator.  Check out the original Fields paper, they didn't have His
selection..  It is strange that your 3-AT isn't working at 200 mM.  That
should kill most strains.  Maybe your strain is screwy.  Try a fresh
isolate.
	  I might be concerned that your bait is providing some trans
activation, since it makes your strain His+, just not enough to turn blue
alone.  Try a mini-library screen on 1000 transformants.  See if an
ungodly number turn blue.  I think you need something less than 1% blues. 
Assume genome size 100,000 ORFS this = 1000 positives.
	You might want to try a less sensitive reporter strain with fewer 
lexAop sites in front of your reporter genes.  Also maybe a library with 
a less potent transcriptional activation domain than VP16 might help.
Good luck,

Mike Moser                                             Tel: 206-543-5354
UW Department of Biochemistry                          FAX: 206-685-1792
Box 357350                                        moser at u.washington.edu
Seattle, WA  98195-7350                     Make peace my beast is yeast

On 22 Oct 1995, Ingrid Wolf wrote:

> I have cloned a plasmid encoding a LexA-fusion protein which I want to 
> use for the yeast two hybrid screen. This bait-plasmid alone or together 
> with the empty VP16 vector allows already growth of the transformed 
> yeast clones on plates lacking His but they stay white on X-gal 
> plates.With a positiv control, a known interacting protein fused to 
> VP16,  the yeast transformants grow on plates lacking His and turn to 
> dark blue on X-gal plates. The background growth of the bait on plates 
> lacking His can not be suppressed by 3-aminotriazole (I titrated all 
> concentrations up to 200 mM). My question is:
>  Has anybody made a two hybrid screen by monitoring the interaction only 
> with the blue color?
> Please give me a message to:      iwolf at fhcrc.org
> Thank you.
> 
> 
> 
> 




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