PCR of pACT2 vector

Greg Cosentino cosentino at medcor.mcgill.ca
Tue Feb 27 16:28:19 EST 1996


Greetings,
I am screening a 2-hybrid library in the pACT2 vector (Elledge lab) and am
trying to get the positive clones out of the yeast and into bacteria. I
have tried the "smash and grab" approach followed by electroporation into
KC8 bacteria with limited success. I am now considering abandoning this
approach and rather PCR amplifying the cDNA inserts out of the "smash and
grab" extracts and subcloning them back into some appropriate vector. Are
there reasons why this shouldn't work? Can anyone suggest decent primers
appropriate for the pACT2 vector for this purpose? I have a good forward
primer (in the GAL4 activation domain) but I'm not sure about a reverse
primer.
Thanks for any help!
Greg
McGill University
Montreal Canada



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