HELP: Yeast 3-hybrid problem

[Bernard Murray, PhD]

In article <3834573400000477 at ems.salk.edu>, mayr at salk.edu 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), I repeated my transformation with a 
> complete new set of reagents and media, still the same result.
> Has anybody got an idea what's wrong ?

I feel obliged to ask the obvious question - does your host strain
grow on -His media without transformation with a plasmid?
(ie. has your yeast stock become contaminated with a HIS strain?)

If so I can only suggest you obtain a fresh isolate.  When you make
your competent cells it is a good idea to see if they behave according
to their listed genotype.  Try a test transformation using a HIS
plasmid and streak the untransformed and the transformed cells on
+/- His media.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF