Ghost bands on plasmid preps

Daniel MacArthur dmac125 at hotmail.com
Sat Mar 31 19:11:18 EST 2001


Dear group,

I'm an undergraduate biochemistry student currently attempting to write up a
practical report on a plasmid miniprep practical. Briefly, we took two E.
coli colonies transfected with pBluescript IISK+ vectors (one containing a
fragment from an EcoRI digest of lambda phage DNA and one with no insert),
purified the plasmids using a Quantum Miniprep from Bio-Rad, digested the
plasmid DNA using EcoRI, BamHI, and a combination of the two, and then ran
out the products on an agarose gel.

The experiment worked beautifully, but I'm having a little trouble with
interpreting the results. In several of the lanes there are faint bands
running at roughly half the size of the full-length linear plasmid, which my
demonstrator called "ghost bands". She told us that these bands were
actually single-stranded plasmid molecules formed during the cell lysis step
of the miniprep, which seems reasonable given their size in relation to the
full-length plasmid. However, we were also referred to two articles by Paul
Hengen (who I believe posts/posted on this newsgroup) which seem to be
putting forward considerably more complex explanations for the existence of
these "ghost bands" (e.g. that the bands represent "double-stranded, cyclic,
coiled DNA composed of two intertwined, but permanently denatured, single
strands of plasmid DNA").

Does anyone have any suggestions regarding the nature of these bands? My
demonstrator's explanation is attractively straightforward and seems to fit
the data with respect to the plasmid without an insert (the linear plasmid
runs at ~3 kb and the ghost band at ~1.5 kb), but it doesn't quite fit with
respect to the plasmid with an insert (linear at ~7.3 kb, ghost at ~2.7 kb),
and seems to be contrary to Paul Hengen's explanations. Any advice on this
matter would be greatly appreciated.


Daniel MacArthur.
3rd Year Medical Science
University of Sydney, Australia





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