yeast plasmid

bong yambao maria at res.agr.hokudai.ac.jp
Thu Jan 24 06:33:21 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.

i had the same problem before until i used sorbitol-zymolyase. worked well with me.




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