Yeast two-hybrid vectors
bmbthc at bmb.leeds.ac.uk
Wed Feb 5 12:24:32 EST 1997
In article <nayers-2101971708270001 at ktraxler.utmem.edu>,
nayers at utmem1.utmem.edu (nancy ayers) wrote:
>.....pGBT9 and pGAD424 vectors. Initially in trying to quantitate the
>beta-gal activity I thought perhaps simply the problem was substrate
>sensitivity, as I can get colonies to grow on triple drop out plates.
Nancy, I've done a bit of work with the same vectors. I don't have any
highly expressing vectors but I do have a suggestion. Which S. cerevisiae
strain are you using? We have three strains, all SUPPOSED to be HIS3
mutants (and therefore unable to grow on His- plates). However, one will grow
very slightly and the other grows like normal on His- plates! Obviously, with
your two vectors present and growth on his- plates, you would expect this to
be due to activation via an interaction, but you get no blue colour when
you assay B-Gal activity. If your yeasts strain(s) do grow on his- plates
when non-transformed, you might want to try 3-amino-1,2,4,-triazole (3-AT).
This chemical inhibits the enzyme (from HIS3 gene). You will need to titrate
the amount required for your strain as this will vary. Try a range from
1mM - 100mM to begin with. What you want is to just inhibit the background
growth and so allow any interaction to produce visible growth.
Hope this helps!
Dept. Biochemistry & Mol. Biol.,
University of Leeds,
email: bmbthc at bmb.leeds.ac.uk
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