Brent/LexA 2-hybrid screen: Gal induction before selection?

S. Findley polliwog at u.washington.edu
Fri Aug 29 01:44:56 EST 1997


Ok...so once a cDNA library has been amplified and frozen down prior to
the actual selection step (i.e. bugs have bait, reporter and a million
different cDNAs and I am about to plate onto HUTL- Gal/Raff plates to
select for interating cDNAs)

The original Brent protocol I think had a short step prior to selection
plating in which the cells were grown for a couple hours in liquid medium
containing Galactose+Raffinse *prior* to the actual plating. But the
Clontech manual (they bought the Brent system and now distribute the kit)
does not include this pre-plating step...

Does anyone know if this pre-plating step is a good thing or a bad thing?


Also...what is a typical period for a weak (or strong) interaction to show
up as a colony at the selection step? 4 days? a week???


Thanks very much in advance!

-Polliwog
-- 
Seth D. Findley               polliwog at u.washington.edu
Department of Biochemistry    Phone: 206-543-1710
Health Sciences Building J579
University of Washington, Box 357350, Seattle, WA 98195



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