Question on generating nicked circular DNA using EthBr and EcoRI
Zophonias O. Jonsson
zjons at vetbio.unizh.ch
Sun Oct 23 13:51:15 EST 1994
In article <3899ge$f31 at news.doit.wisc.edu>, Rock Pulak
<rapulak at macc.wisc.edu> 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.
A similar procedure for generating random nicks using DNaseI is described
Takahashi M., et al. (1984) J. Biol. Chem 259:10041-10047
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