Brent/LexA two-hybrid system

Helen McBride helen.mcbride at genetics.utah.edu
Fri Sep 5 16:13:41 EST 1997


Dear Seth,
    The Galactose in the medium is to induce expression of the prey gene
that is under the control of the Gal1/10 promoter. Inducing before
plating really doesn't make a difference as you will be plating on plates
containing galactose. That is probably why it was eliminated from the
protocol. But induction won't hurt if you wanted to use the original
protocol, either. 
   Since you are selecting for three plasmids as well as the reporter
product from one of those plasmids, give the yeast an extra day than
normal to grow up. So that would mean four days growth would probably be
the first positives. However, when the original transformants grow up
really isn't the best sign of the strength of the interaction as other
factors may be involved in how large the colonies are: for example, how
sick the overexpressed prey makes the cells. Even titrating with 3-AT
doesn't always indicate how strong the interaction is, although it is
just as good as looking at the back-up check of a lacZ reporter. Good
luck in your screen.
Helen McBride
University of Utah 
Grad Student
Dept. of Onc. Sci.
helen.mcbride at genetics.utah.edu


"Where the telescope ends, the microscope begins, which of the two has
the grander view?" Victor Hugo



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