Dave,
Try using SD-leu medium with 0.05% 5-FOA. 0.1% might be too much. Also
make sure your -leu medium has uridine or uracil. The cells need uridine
or uracil in order to grow without the URA3 gene.
There is also a paper which describes the use of proline as the nitrogen
source during 5-FOA selection. This allows one to use a lot less 5-FOA
McCusker and Davis (1991), YEAST 7(6):607
Hope this helps,
Mike
In article <8nkekt$13h$1 at mercury.hgmp.mrc.ac.uk>,
Dave Lloyd <djl17 at leicester.ac.uk> wrote:
>I have conducted a two hybrid screen and have a selection of positives.
>Bait plasmid's marker is URA3, and Library plasmid's marker is LEU. Now i
>want to lose the bait plasmid to plasmid rescue the library plasmid, and
>also to check that this plasmid is not self-activating. I pick a colony
>and grow in SD-LEU media till saturation then streak out on SD-Leu +
>0.1%FOA plates, but get nothing back, why is this??
>I am attempting to do two rounds of culturin before i streak out on the FOA
>plates. Or i could spin down my cultures and resuspend in a small volume
>and plate it all out on FOA plates however this would mean having one FOA
>plate per culture (or Y2H positive) thus a lot of plates, and lots of
>money....
>>************************
>Dave Lloyd
>Division of Medical Genetics
>University of Leicester
>Leicester
>LE1 7RH
>UK
>Tel: +44 116 252 5088
>Fax: +44 116 252 3378
>************************
>>>---
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