Ghost bands on plasmid preps

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Sat Mar 31 20:10:05 EST 2001


Hmmm... without the benefit of the snapshot of an actual digest, it is
difficult to interpret any such results. However, the explanation of
"single stranded" plasmid (if that is what your demonstrator really meant)
is not correct. single stranded nucleic acids do not run as a tight band
and certainly not at 1500bp size on a TAE or TBE gel.

The first possibility that comes to mind is that what you are observing in
the gel are "un-digested" supercoiled plasmid. This is rare, since after
overnight digestion , especially, it would be hard for any plasmid to exist
in supercoilded form. 

BTW, did you run the undigested plasmid, with and without the insert,
alongside? If you did, how did those lanes compare? If you didn't, you should.

The second possibility: Although you have not described in great detail, is
it possible that the EcoRI insert from the phage DNA contains a BamHI site?
>From what you have said, the insert is ~4kb. So, think in terms of an extra
BamHI site. Does the BamHI digest (single) produce any band smaller than
what is produced by single EcoRI digest?

I don't remember what Dr. Hengen had explained about "ghost bands", but I
think he was talking about undigested samples. It is a common observation
that even when a supercoiled plasmid is excised from a gel and
electrophoresed, it resolves into  a relaxed form and a supercoiled form.
This phenomenon is dependent on the buffer, the gel, the gel %, the plasmid
prep, voltage applied, etc. 


Hiranya

At 10:11 AM 4/1/01 +1000, Daniel MacArthur wrote:
>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
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu

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