yeast plasmid

SLF notmyaddress at hotmail.com
Wed Jan 23 11:21:55 EST 2002


>
>
> Dear yeasters,
> We have some problems with DNA isolation of yeast. For some reasons, I
> obtain little DNA, however, after subsequently transforming E.coli, in hope
> to recover our desired plasmid, we never yield colonies on the selectable
> carbenicilline (for the plasmid) plates. In addition, we didnot succeed
> either with the clontech-kit for yeast plasmid isolation.
> My question is: is this a common problem that occurs particularly when
> isolating DNA form yeast, and how can we overcome the problems. Is good
> enzym treatment better than mechanical disruption of yeast cell walls? We
> also tried regular purification of the DNA, however, with no substantial
> differences.
> Hope you guys can help me out with this.
> Thanks so much


This is a common problem.  It appears that there are various inhibitors of E. coli
transformation in  yeast preps.  We found that the best way around it was to
transform coli by electroporation, rather than by normal CaCl competent cells.
While not great, it has enhanced our recovery.


--
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