plasmid rescue experiments

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Article #29137

#29137

From: Department of Molecular Karma
Date: Thu, 24 May 1990 17:59

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I'm experiencing a problem with a plasmid rescue experiment. It appears
that the overwhelming amount of host cell DNA isolated in the total DNA
preparation effectively masks most of the rescuable plasmids when
transforming E. coli DH10B (BRL).  I am sure that this is occurring
based on experiments in which I dose the total DNA preparation with some
control plasmid before transformation.  These control plasmids are also
masked. The effect seems to be due to the fact that relatively
high amounts (10s of ng ) of total DNA must be used in transformation
in order to see any rescue at all.

I have tried further purification of the total DNA preparation
via spun-columns and see no relief of this effect. I am considering
trying to eliminate the linear DNA from the total DNA preparation
via exonuclease or alkaline treatment. I thought that I would post
this query first hoping for some good advice before I waste some of
these DNA preparations. If anyone has any experience in plasmid rescue
or some other enlightening comments please e-mail me directly.

Thank you,

 Michael D. Yablonsky                  internet: yablonsky@biovax.rutgers.edu
 Waksman Institute                     at&tnet: (201) 932-3052
 Rutgers University
 Piscataway, NJ, 08854-0759

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From: YABLONSKY@BIOVAX.RUTGERS.EDU (Department of Molecular Karma)
Newsgroups: bionet.general
Subject: plasmid rescue experiments
Message-ID: <9005241803.AA06670@genbank.bio.net>
Date: 24 May 90 17:59:00 GMT
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Posted: Thu May 24 18:59:00 1990