Store DNA concentrated or dilute?

Bernard Murray bernard at elsie.nci.nih.gov
Tue Mar 12 15:49:48 EST 1996


In article <4i1hve$59m at news.duke.edu>, vando006 at mc.duke.edu says...
>
>Bernard,

[SNIP]

>(I do not understand the concatamer formation).
>
>Margon
>

I should clarify my own position on this.  I was simply passing on
a non-net-user's opinion on DNA storage and I myself have formed no
particular opinions on the possibilities of plasmid concatamer
formation at high DNA concentrations.  I'll pass everyone's comments
on to the person concerned.  The reason that I give her credence is
that she is excellent at transient transfection and I tend to
follow her protocols/recipes/cookery with some trust.  As I indicated
in my earlier post I simply dilute my plasmid preparations to a
nominal concentration (eg. 1 g/l) to make the calculations easier
(rather than to prevent any rearrangement events).  I shall quiz the
person concerned about the concatenation.  I think I originally
rationalised it as an extension of the spontaneous degradation of
DNA in acidic (or unbuffered) solutions in that at high concentrations
of DNA the low concentration of Tris in most TE (eg. 10 mM) would
be insufficient to protect from this.  Of course, true concatamer
formation would require the reannealing of the chains that broke
to allow the interlinking to take place.  I certainly don't believe
that you get;
2 x separate supercoiled plasmids -> 1 x supercoiled concatamer
(at least in the absence of any enzymes).

In future I'll ponder other people's claims a little longer before
posting them in public.  No, on second thoughts, how else will
preconceptions be destroyed (eg. the famous "ethidium bromide is
dangerous" story).

		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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