HELP: Yeast 3-hybrid problem

PMBie lab pmb at auckland.ac.nz
Sun Nov 21 18:32:41 EST 1999


I haven't done 3-hybrid, but am currently doing two-hybrid. Have you
thought of trying the strain PJ69-4a? It has much lower background growth
on -his media than Y190. For example you generally don't need any (or 0-5
mM) 3-AT.

cheers
Kim

In article <spam-1911991944470001 at macmac-2.ucsf.edu>, spam at 127.0.0.1
(Bernard Murray, PhD) wrote:

> 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.




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