Yeast 2-Hybrid Screening

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Wed Nov 11 13:00:10 EST 1998


Martin Cann wrote:
> 
> I'm working on the yeast 2-hybrid system and have some questions.  After
> triple selection (-Leu/-Trp/-His), I have hundreds of colonies (>300).
> I am currently doing a blue/white screen.  Does anyone have any
> suggestions for cutting down the number of colonies.  I do not think
> that these are all "ture" poitives and would like to cut down the number
> significantly before I start to purify DNA and sequence it.
> 
> Barbara

Dear Barbara,

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.
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.
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.

If I can be of any further help, don't hesistate to contact me.
Cheers
Ricky



******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de



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