Plasmid rescue from S.cerevisiae

paul bundock bundock at rulsfb.leidenuniv.nl
Wed Jan 10 17:22:40 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.




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