nicking DNA plasmids on purpose

Tim Barnett t.barnett at path.utas.edu.au
Wed Feb 28 18:46:48 EST 1996


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. I have tried using DNase I, but have found that
> after the first nick, the second cut is always at the nick site. So
> instead of multiple nicks, I am producing linear DNA. This is using
> both Mg and Etbr.
> 
> Does anyone have any ideas? Apart from just leaving the DNA for a few
> years in the fridge that is.
> 
> Thanks.

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.  I don't know what you should put your plasmids in (since agarose would probably be less than ideal), but I guess dialysis membrane might work.

-- 
Tim Barnett                             fax:   61-02-354833
Department of Pathology                 phone: 61-02-354825
University of Tasmania                  email: t.barnett at path.utas.edu.au
Hobart, Tasmania 7000
AUSTRALIA



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