Bulk prep problems (Yes, plasmids can integrate)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Oct 31 15:43:08 EST 1994


biocmag at sanger.otago.ac.nz (Michael Gieseg) writes:

> So it appears that even though the bugs are ampicillin resistant they conatain
> no plasmid.  Is it possible that the plasmid has integrated?

I once screened pBR322 transformants directly by Southern blot and picked up a
couple of colonies wherein the pBR had integrated.  So it does happen at some
rate.  More commonly though, people just don't replate enough to get rid of 
feeders, and then drop the selection and let the feeders overgrow the 
transformants.  I've also seen experiments that were just contaminated with
some extraneous bug that was naturally amp resistant.  I do two things that 
generally excludes misbehaving clones from my experiments.  1) I replate
all colonies 2 x before conducting any screening beyond the drug resistance
and the lacZ color test.  This lets anything that's going to be unstable
in any way disappear before I spend any effort on it.  2)  I do some kind 
of miniprep (often just a toothpick assay) to verify correct size and good
yield of plasmid from a clone before going to the large prep.  I think this
probably screens out weak producers of various kinds.

Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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