high molecular plasmid DNA

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Fri Jan 11 22:52:25 EST 2008


In <mailman.153.1200074433.2451.methods from net.bio.net>,
 Dr. Hiranya S. Roychowdhury <hroychow from nmsu.edu> wrote:

> Well, this has never been suficiently explained, nor do I know of any
> specific investigation into this.  I tend to believe that the high MW
> species may exist as a complex of bacterial chromosome and other proteins.
>  Usually, a clean plasmid prep does not show the HMW bands.  It also has
> nothing to do with the conc. of the agarose gel.

Excessive treatment with alkali will cause this to appear.  When nicked
DNA denatures, it creates a fast-migrating single-stranded circle that
is "undigestable" by restriction endonucleases.  The other product is
linear ssDNA that tends to aggregate (by partial base-pairing) into 
large complexes that migrates near the well of agarose gels.

If you treat minipreps with a single-strand specific nuclease like
S1 or mung-bean nuclease both aberrant species (hmw ssDNA, and fast-
migrating ssDNA) will disappear.

AC
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