nicking DNA plasmids on purpose

Ned Mantei mantei at neuro.biol.ethz.ch
Thu Feb 29 05:20:23 EST 1996


In article <t.barnett-2902961047490001 at 131.217.101.125>,
t.barnett at path.utas.edu.au (Tim Barnett) wrote:

> In article <DnHEEI.Dzz at uns.bris.ac.uk>, vipond at bsa.bris.ac.uk wrote:
> 
> > I am trying to produce a multiply-nicked DNA plasmid for some assays,
> > but am having trouble.
> In Southerns you incubate gels in 0.25M HCl, for 20min or so, which
depurinates the DNA.  You could try incubating your plasmids for varying
times in this solution and then neutralise in Tris buffer.

The acid merely depurinates, but strand breakage doesn't occur until the
DNA is exposed to alkali. Depurinated DNA has alkali sensitivity similar
to that of RNA--it takes awhile or some heating in NaOH to get strand
breakage at every depurinated site.
One can control the degree of depurination by using less drastic
conditions than 0.25 M HCl: Incubation for 20 minutes at 55 C in pH 4.5
acetate buffer will produce about 1 depurination per 5 kb (Within a factor
of several--it's been 20 years since I did these experiments. There was a
paper on the subject in the late 1960's or early 70's, from a Swedish
group and probably in Biochemistry.)

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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