Library plasmid rescue in two-hybrid sysytem

gmclab221 nayers at cctr.umkc.edu
Thu Aug 13 12:39:15 EST 1998


Hello all. I have been trying to retrieve  positive prey plasmids from
yeast EGY48 after a two-hybrid library screen. I am attempting to
electroporate KC8 bacteria which are -trp deficient with a yeast DNA
mini-prep in order to rescue the plasmid but I cannot get any
transformants. I have checked my elctrocompetent cells with a control
plasmid and they are fine. I also am plating on less stringent LB/Amp+
plates initially instead of minimal media plates in order to improve my
chances but still no transformants. I am thinking that maybe the plasmid
concentration from the mini-prep is insufficient or too dirty for
electroporation? I am phenol:chloroform extracting the mini, however. I
was just wondering if anyone else has had the same difficulty in
rescuing their preys in KC8 and would greatly appreciate any suggestions
or advice!
Thanks so much,
Nancy

nayers at cctr.umkc.edu
School of Biological Sciences
University of Missouri-Kansas City
5007 Rockhill Road
Kansas City, MO 64110



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