plasmid purify from yeast for transformation

AShaw5 ashaw5 at aol.com
Sun Jan 29 22:23:58 EST 1995


This is a difficult problem.  Cleaning up the DNAs using wizard kits we
were told would help but didn't.  We have tried various methods.  Using
library efficiency bugs and electroporation, we usually get most of them
transformed on the first round.  Repeated tries for the others eventually
gets all of them.  We have also tried PCR with success.  Inserts can be
sequenced directly, or restricted and then ligated into vectors.  Lastly,
we have tried making fresh competent bugs.  That seems to have worked best
for us with great numbers of colonies.  I wonder whether the DMSO in the
frozen competent bugs is inhibiting?  Anyone out there with similar
thoughts.
     I hope that you have kicked out your bait plasmid already.  If you
haven't you will end up transforming both plasmids.  We have also tried
transforming HB101 bugs on minimal plates to take advantage of the leu
selectable marker.  That was tough but works on occasion.

good luck
Andrey Shaw



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