background in 2 hybrid system
moser at U.WASHINGTON.EDU
Thu Oct 26 11:36:29 EST 1995
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
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.
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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