high molecular plasmid DNA

Dr. Hiranya S. Roychowdhury via methods%40net.bio.net (by hroychow from nmsu.edu)
Sat Jan 12 19:02:30 EST 2008


This is a perfectly good explanation.  Thank you. Nicking of the plasmid
-as well as of the chromosomal pieces - does cause the HMW bands to
appear.  Another interesting observation is that often a clean and
perfectly good prep will show these spurious species on subsequent
electrophoresis (especially following storage).

> 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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-- 
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003



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