Question on generating nicked circular DNA using EthBr and EcoRI

Zophonias O. Jonsson zjons at
Sun Oct 23 13:51:15 EST 1994

In article <3899ge$f31 at>, Rock Pulak
<rapulak at> wrote:

> Does anyone have a protocol for generating nicked circular DNA from
> supercoiled DNA by using ethidium bromide and Eco RI (or any other 
> restriction enzyme)?  Either a detailed protocol or a reference would
> be greatly appreciated.  

It's simple, just add saturating amounts of EtdBr to your restriction mix
(I use 20 ug/ml) and incubate.  You must experiment a bit to find the
aproppriate ratio of enzyme and template.

Take a look at:

Barzilai, R. (1973) SV40 DNA: Quantitative conversion of closed circular to
open circular form by an ethidium bromide-restricted endonuclease. J mol.
biol. 74:739-742

A similar procedure for generating random nicks using DNaseI is described

Takahashi M., et al. (1984) J. Biol. Chem 259:10041-10047

Good luck


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