HELP: Yeast 3-hybrid problem

Rafael Maldonado rmaldonado at ua.es
Mon Nov 22 10:50:08 EST 1999



Bernhard Mayr wrote:
> I'm trying to do a yeast 3-hybrid screen using Clontech's pBridge and
> pACT2 vectors. My problem is, that whatever vectors I transform the
> yeast always grows on -His media, even when I transform empty pBridge
> and pACT2 vectors and rise 3-AT to > 200mM. The yeast wt-phenotype is
> OK (tried CG1945 and Y190)

Hi Bernhard,

This is a stupid question, but did you test the three phenotypes (his-,
leu- and trp-) separately (I mean, medium -his +leu +trp)? Could have
you done some mistake in the media composition, i.e., adding histidine? 

Also, there is a set of yeast strains which use the ade2 gene as
selection system, which is much cleaner than his3 selection (no need of
inhibitors). Besides, the his system can be used as a different control
for interaction. It works great in our hands, although you could lose
some weak interactions.


-Rafa

-- 
Rafael Maldonado
Divison of Genetics
University of Alicante (Spain)




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