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Library plasmid rescue in two-hybrid sysytem

S. Simon renniesl at ?cc.umanitoba.ca?
Wed Aug 19 16:10:36 EST 1998


In article <6qv8c0$lhq at net.bio.net>, gmclab221 <nayers at cctr.umkc.edu> wrote:

> 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


Hi Nancy
   We are using a different 2HS than you but recover our positives (LEU)
in KC8 as well
We have found that plating on LBamp and replica-plating to M9-leu works
well to recover
the positives..... so What method are you using to extract the DNA from
your yeast??  Maybe this isn't 
giving you the conc'ns needed?  We traditionally use Charlie Hoffman's
glass bead preps (Hoffman and Winston Gene 57: 267-272)   Quick easy and
effective.  We also have an older longer extraction using zymolyase that
we use for recovering genomic DNA for Southern's (Cryer et. al. Methods
Cell Bio 1975 12:39-44),  it does give higher nucleic acid yields, but I
don't think it would be necessary?
   
Hope this gives some insight
Sharon

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