plasmid rescue from...

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Wed Jan 10 11:32:33 EST 1996


>I am trying to rescue a chromosomally integrated plasmid from
>S.cerevisiae by plasmid rescue.Total DNA is isolated from the
>transformants and digested with BglII, ligated in a large volume
>(300ul),ppt, and transformed to E.coli strain MC1061 by electroporation.
>I am encountering difficulties as the plasmid carries some direct
>repeats, so after rescue to MC1061 (recA+) I get the same recombination
>product in the MC1061 colonies.I have tried rescueing to DH5 alpha
>(recA-), and still get the same recombination product.Does anybody have
>experience with this and could suggest alternative recombination
>defiecient E.coli strains I could try? Thanks, Paul B.           

Hello Paul,
I would suspect recombination happening in yeast before
DNA purification.
Regards
Andrei




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