re-iterating positives

Barry Young byoung at plato.wadham.ox.ac.uk
Mon Mar 6 17:45:34 EST 2000


On 5 Feb 2000 11:05:26 -0000, lindus conlan <l.conlan at pgrad.unimelb.edu.au> 
wrote:
>Hello,
>I've just completed two library screens using the Matchmaker One system but
>using pJ-69 4A and have obtained a number of putative positive
>interactors.After obtaining the plasmid DNA from the yeast, transforming
>into KC8 cells for mini-prep,and then re-transforming back 
>into the yeast with my bait, I am no longer able to 
>re-iterate the majority of the positives. The plasmids contain inserts. Any
>advice would be greatly appreciated.

Unfortunately, PJ69-4A has a habit of throwing up quite a lot of non-specficic
positives (i.e. activating reporters regardless of bait/prey). As I've said in
a previous post, you can screen a lot of these out at an early stage by
streaking on to FOA-Ade, and discarding anything that grows.

Another possibility, depending on the conditions of your initial library
transformation, is that your positives contained multiple preys. If you go
back to your E. coli transformants (from the yeast DNA extracts) and miniprep
say, 6 of these, you might find a couple of different plasmids.

Hope this helps

Barry

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