Dear yeast community:
I have been using the yeast two hybrid system to search for proteins
which interact with a plant transcription factor (using Stratagene's
Hybri-ZAP system). I have gotten several positives which behave oddly
(at least to me). Can anyone offer advice on the following:
1. A positive clone was picked and plasmid DNA prepared from it. This
DNA (prey plasmid) was used to transform yeast harboring the bait
plasmid, and transformants selected on -trp (bait) and -leu (prey)
medium. If ten colonies from this plate are then plated to triple drop
out (TDO) medium, only some grow. All seem to have both plasmids, and
thus should be genetically identical. If the plasmids were integrating,
I could rationalize that some were inserted into transcriptionally
silent regions and therefore would not be able to grow on the TDO, but
it is my impression that the plasmids are non-integrating. Does anyone
else see such things and/or have an explanation for me?
2. I have some positives which grow on TDO, but not all pass the false
positive test (ie give rise to positive b-galactosidase test by the
x-gal filter lift assay). Most of those which do give rise to color are
not very convincingly positive. That is, it takes 15 hours or so to
develop color and the color intensity is weak. (Positive controls give
strong color reaction in less than 6 hours). Other colony grids remain
white. I guess what I am asking is "what is the threshold at which one
deems this second test to be valid?" Do other people see weak color
reactions? Is there another way to be more convinced that there is
truly a positive interaction?
Thank you for your help.
--
Dan Riggs
Botany Department
University of Toronto
1265 Military Trail
Toronto, Ontario M1C 1A4
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