Here's what we do:
5 tubes with serial dilutions of enzyme:
Tube 1 2 3 4 5
plasmid 6ug 4ug 4ug 4ug 2ug
enzyme 1ul 0 0 0 0
total vol. 30ul 20ul 20ul 20ul 10ul
The amount of enzyme in the 1st tube is what is neede for digestion
in one hour. THe ratio of the ingredients inthe 5 tubes are the
same except for the emzyme.
After adding the enzyme to tube 1, mixing it in well, I transfer 10 ul from
tube 1 to tube 2. Then 10 ul from tube 2 to tube 3 etc. till tube 5. So I
end up with the same amount of DNA in each tube but serial dilution of the
enzyme. Then I let the reaction go half hour and immediately stop it (which
I do by adding excess of EDTA and phenol/chloroform).
In the best case this results in a range with the first tube being fully
digested and the last tube totally undigested. And somewhere in the middle
you will find the fragment you are looking for. But even in less ideal cases
I would always have one or two tubes with incomplete digests that gave me the
Thon de Boer (deboer at nefeli.imbb.forth.gr) wrote:
: 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)