Thon de Boer
deboer at nefeli.imbb.forth.gr
Sun Feb 23 17:56:26 EST 1997
In article <5dsv3d$k1b at mserv1.dl.ac.uk>, Daniel Auerbach
<auerbach at cell.biol.ethz.ch> wrote:
> Hi everyone,
> I'm trying to do a limited digest with Nsi I on a 5.5 kb plasmid. I first
> cut with Eco 47 III and then add Nsi I. The aim would be that Nsi I cuts
> only once, so that I can isolate the corresponding band. Unfortunately,
> with every condition I try, Nsi seems to cut several times immediately (I
> have 3 Nsi sites in the plasmid) resulting in a smear of bands and nothing
> for me to isolate!
> Can anybody tell me if there are conditions which influence an enzyme to
> cut only once ? I tried low amounts, wrong buffer and lower temperatures,
> nothing seems to work.
I found a method for limited digestion using Etidium bromide in Maniatis.
Et. Br. intercalated into the DNA inhibits restriction. Supercolied DNA
doesn't contain much Et. Br. when compared to relaxed lineair DNA so the
first cut is rather efficient, but when the cut DNA relaxes Et.Br. can
enter the DNA and inhibits further digestion.
First do a pilot with Et.Br. concentrations between 20-60 microgr/ml and a
time course of those(0-30 min), since every enzymes reacts differently.
This worked great in my hands, using NcoI.
Et.Br. also apparently lifts the site-preferences some enzymes have so you
could end up with a good mixture of DNA cut only once.
So first do the limited NSiI restriction, clean it up and cut with the Eco47III.
Hope it works (let me know, please)
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