"Ghost" bands in plasmid preps?

[Bernard Murray, PhD]

In article <383C5889.ED006DEB at cc.umanitoba.ca>, Rob Kirkpatrick
<kirkpat at cc.umanitoba.ca> wrote:

> Simon Dawson wrote:
> 
> > Hi all,
> >         Does anyone ever see what I can only call "ghost' bands if they
> > over-expose an ethidium bromide stained agarose gel of plasmid DNA? They
> > usually appear to run somewhat faster than what I would think is the
> > supercoiled DNA band and you only seem to see them if you overexpose the
> > gel.
> >     Any thoughts? Ignore them (they don't seem to cause any problems with
> > subsequent manipulations)? I'm curious to know if anyone else sees something
> > like it.

> I'm told these are random cleaved DNA fragments that result
> from excess exposure
> to NaOH in the lysis step and that they are resistant to endonuclease
> cleavage.
> But, I'm am not an expert on this and am somewhat interested in the answers to
> this question as well.

Paul Hengen tells the story in his March 1994 TIBS column and
the text is available here;
    ftp://ftp.ncifcrf.gov/pub/methods/TIBS/mar94.txt

The ghost band usually runs about the same MW as ssDNA of
"non-denatured" plasmid.  I don't believe "random cleavage"
is involved.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF