Hi Dave:
An alternative way to get your LEU plasmid is this: Do
plasmid rescue on your original strain containing both
plasmids, but for transformation use a bacterial strain that
is leu-. Plate in a single colony density onto LB+ampicilin
plates, and make replica on a M9 plate (Maniatis/Molecular
Cloning). If the bacterial strain has other auxotrophic
requirements don't forget to supplement the M9 medium as
required (for example I used JA221 which is also trp-).
Cheers,
Martin
Dave Lloyd 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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