Yeast 2-Hybrid Screening

[Barbara Gorfajn]

Ricky,


I have a few more questions, and some answers for your questions.....


do you include any inhibitor of basal HIS3 expression, like
3-aminotriazole
into the medium? Which reporter strain do you use? Different reporter
strains
display different signal to noise ratios depending on the promotor which 
controls reporter gene expression.



I have not included 3-AT.  When I tested the yeast transformed with only
bait plasmid on a His- plate, I did not get much background and figured
I could go on with out 3-AT.  I am using yeast strain CG-1495.



Why do you think your His+ are not LacZ+ before you tested them? If you
have
a kind of very "sticky" bait protein I would think 300 positives could
be 
true. I experienced a great variation between His+ and LacZ+ depending
on
the bait protein I use. Sometimes between 80 and 90% of the His+ also
show
lacZ expression, in other cases I get tons of His+ and none of those
turns blue
when tested.


The bait I am currently testing is probably very sticky.  It contains a
potential leucine zipper.  I am expecting to have a large amount of
false positives.  I am currently doing the blue/white selection.  I have
chosen to use xgal plates.  I am worried that this method may not be
sensitive enough for the yeast strain that iI am using (CG-1495).  I may
switch to the filter lift assay.  Do you have any advice in this area. 
I have plates about 200 colonies on xgal plates already, and after 2.5
days, none of the colonies have turned blue.



Before you start to sequence a huge amount of putative positives you
should do
the respective control transformations like transforming the isolated
library
plasmids back into the reporter strain harbouring the original bait
plasmid and
into a strain harbouring a functionally unrelated hybrid-protein. In my
experience 
the positve phenotype is not reproduceable in all cases when the target
is 
transformed back into the original strain.


I have planned on doing this

Thank-you

Barbara
bdgorfaj at mail.med.cornell.edu