HELP: Yeast 3-hybrid problem
Bernard Murray, PhD
spam at 127.0.0.1
Fri Nov 19 21:44:47 EST 1999
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 P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
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