Two hybrid advice?

Barry Young byoung at plato.wadham.ox.ac.uk
Fri Mar 17 11:27:56 EST 2000


On 11 Mar 2000 23:33:00 -0000, Dan Riggs <riggs at scar.utoronto.ca> wrote:

>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?

It's not clear from what you've said, but I assume that you transformed
the recovered library plasmids into E. coli first. It might be the case
that your original positive clone contained multiple preys, only one
of which is causing the interaction. I'd check maybe 6 E. coli
transformants, do a diagnostic digest, and make sure that they're all
the same.

Barry

-- 
Barry Young
School of Biological Sciences
University of Manchester
Manchester
UK
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