Yeast 2-Hybrid Screening

Barbara Gorfajn bdgorfaj at MED.CORNELL.EDU
Wed Nov 11 10:29:58 EST 1998


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

do you include any inhibitor of basal HIS3 expression, like
into the medium? Which reporter strain do you use? Different reporter
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
a kind of very "sticky" bait protein I would think 300 positives could
true. I experienced a great variation between His+ and LacZ+ depending
the bait protein I use. Sometimes between 80 and 90% of the His+ also
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
plasmids back into the reporter strain harbouring the original bait
plasmid and
into a strain harbouring a functionally unrelated hybrid-protein. In my
the positve phenotype is not reproduceable in all cases when the target
transformed back into the original strain.

I have planned on doing this


bdgorfaj at

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