Ghost bands on plasmid preps

a.krimer at biosidus.com.ar a.krimer at biosidus.com.ar
Tue Apr 3 16:10:13 EST 2001


Hi Daniel:

Did you perform Phenol-chloroform extraction on your minipreps?
Some years ago I have had the same problem and I solved the problem
shorting the SDS-NaOH step (1-2 min.) and using Phe-chlorof (I didn't
routinely use Phe in my preps)
Hope this helps
Sincerely

Alejandro Krimer




"Daniel MacArthur" <dmac125 at hotmail.com>@hgmp.mrc.ac.uk on 04/03/2001
06:08:38 PM

Sent by:  owner-methods at hgmp.mrc.ac.uk


To:   methods at hgmp.mrc.ac.uk
cc:
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Subject:  Re: Ghost bands on plasmid preps


Hi Rusty,

You said:

> I have experienced something similar in the past, and I might be able to
help.
> First, I don't know that the bands you are seeing are ssDNA, as EtBr
> intercalates very inefficiently to ssDNA, and whatever EtBr that does
> intercalate is thought to be the result of duplex DNA formation.  You can
test
> this ssDNA theory by running some of the undigested plasmid prep on a gel
next
> to the Bam/RI cut DNA-- if there's not a band in the uncut DNA that
corresponds
> to the digested DNA lane, then you know it is something that has occured
during
> the restriction digest.

I did run a lane of undigested plasmid along with the various digested
samples in the original experiment (sorry, I wasn't very clear about this
in
my post). The ghost band is present at essentially identical strength in
all
samples, both digested and undigested. To me this suggests two things: (1)
the problem is with some step prior to the restriction digest, and (2) the
ghost bands (whatever they are) are resistant to both EcoRI and BamHI
digestion (I used both enzymes, alone and in tandem). Perhaps the
restriction sites are absent, or there are regions of ssDNA around the
recognition sequences, or the conformation of the DNA is too tight to
permit
access by enzymes. But what this all says about the identity of the bands I
have no idea. :-)

Your point about EtBr's low (absent?) affinity for ssDNA is an excellent
one, and definitely suggests that the ghost bands are not simply
single-stranded plasmid molecules denatured during the cell lysis stage of
purification.

> You should call the company that makes the enzymes and
> see if any complaints have been logged for the EcoRI and BamHI.  A few
months
> ago we had a similar problem with EcoRI from Promega. They sent us a new
batch
> and the problem went away.  The extra bands may be due to inappropriate
cleavage
> of your plasmid (called "starring").

Apparently the ghost bands appear routinely on gels following the
purification of this particular plasmid (pBluescript), regardless of the
restriction enzymes used, so I don't think it could be contamination of the
enzymes (although that's certainly not out of the question in this
particular case).

Out of interest, do you happen to know what caused the "starring"? Was the
batch contaminated with another restriction enzyme, or was there something
affecting the activity of the EcoRI itself?

> One last suggestion: the migration of  DNA thru the gel is effected by
the
salt
> concentration of the sample prior to loading in the gel.  I know that
when
I
> elute purified linear DNA in H2O, I often get smaller bands than
expected.
I
> can correct the problem by adding 1x of any restriction enzyme buffer and
> letting the sample sit for 2-3min on the bench before loading on the gel.

I suspect that the salt balance was a bit awry as my bands were not as
clean
as I would have liked. However, I'm not sure if salt balance could be used
to explain the ghost bands, as the expected bands for linear, circular and
supercoiled plasmid DNA were all present at roughly the right positions on
the gel, while the ghost bands consistently appear in every
plasmid-containing lane as a faint, fast-running band. Could the salt
concentrations have produced this, or would a salt problem have affected
the
running of all of the bands equally?

I've written your suggestion down, though, as I suspect it may come in
handy
at some time in the future when my gels start giving different sorts of
strange results. :-)

> Good luck.

Thanks!


Daniel.





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