Plasmid question

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Jul 27 16:25:28 EST 2000


Hi Mark,
Ligation products do not have supercoils, but are relaxed circles. They
will therefore not run like formI-DNA (covalently closed supercoiled
circle), but slower. 
HOWEVER, if you have a standard ligation (one used for cloning
purposes), it will be VERY hard if not impossible to see the correct
product. Rather, there will be a lot of bands. Dependent on the termini
of the fragments, you will see linear dimers or multimers of vector, of
insert, and combinations of vector and inserts. In addition, you will
see circularized molecules, but again they can be dimers or multimers of
any fragments.

Transformation into bacteria is a powerful selection for the "right"
constructs, as linear DNA will be degraded rapidly, insert dimers will
not replicate because they have no origin of replication (and no
resistance either...), and vector multimers have problems in
replication, resulting in a failure to propagate. As a result, only
plasmid monomers with or without insert can give rise to colonies. And
still there is much that can go wrong in cloning :) 
Seeing only two kinds of outcome in the minipreps (religated vector
background and vector+insert) has lead to the common misconception that
only these two kinds of DNA are present after ligation. But, as outlined
above, that is not true at all, and I have seen many people who thought
they could  improve the ratio of positive clones over background by
"simply" gel-purifying the "good" ligation product. Needless to say,
they usually gave up.

Hope this helps,
Frank





Mark Garry Petten wrote:
> 
> I am running a sample of plasmid I have ligated with my cDNA insert
> through a 1% TBE gel. I am also running with it a sample of linearized
> plasmid without the insert. My question is will supercoiling cause the
> ligated plamid to migrate less through the gel or more?
> 
> *************************************************************************
> *Mark G. Petten                      * Research is the process of going *
> *Dept. of Biology                    * up alley's to see if they're     *
> *Honours                             * blind.                           *
> *Memorial University of Newfoundland * - Bates's Law of Research        *
> *************************************************************************






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