two-hybrid

[Riekeltje Koedood]

Here's what I did in a similar case. Before lib. transformation it seemed like
as little as 5mM 3AT is sufficient to inhibit growth on His- plates. After 
lib. transformation, I got too many colonies (lots and lots). Repeated the
screen at 35 mM 3AT with smae effect. However, I did let my real screen plates
grow 5 days i.s.o. the 3 days for the 3AT titration exp. 

What I did? I picked all the colonies that seem 'bigger'. In one screen that
was about 125 in the other about 250 colonies. OUt of these I had a total of
4 that turned blue in the b-galactosidase assay. Thus my numbers compared 
very well to those of some of the example 2-hybrid studies I had at the time.

Alternative is to do the b-galactosidase assay directly on the plates after
lib. transformation. However, I was too scared of not bneing able to salvage 
positive clones.

I may repeat my screens for more clones. My 4 positives turned out to be only
2 independent genes.

Good luck.

Marieke

nancy m. dougherty (dougherty at a1.mscf.upenn.edu) wrote:
: We are also having this problem but with the one-hybrid system.  Yeast
: (YWAM2) harboring our CEN reporter plasmid are not his+, at least when
: streaked out on his- media.  When we transform with our library, however,
: we get thousands of his+ colonies.  Replica plating the transformants to
: 150mM 3AT retarded the growth of all of the colonies, but did not reduce
: the number of his+ colonies.  Being rather new to this, we are not sure
: how to get around this problem.  Would changing strains help?  How does
: 3AT inhibit background his expression anyway?
: Any advice you could give would be appreciated.  Sorry we can't help you out!!


:    Please reply to:
:       kucharck at mail.med.upenn.edu