Sequential restriction digest

[Nikola Wenta]

Dear all,
I want to do a SacI/SalI digest of my plasmid in order to subclone my
insert (1.2 kb) into another vector. According to NEB, the REs need
different buffers, so I would have to do a sequential digest. Does
anyone have a working protocol for sequential digests? Particularly I
would like to know how to proceed once the first digest i.e. with SacI
has linearized my original plasmid. Would you run a preparative agarose
gel and cut the band out, or would you precipitate the DNA and resuspend
the resulting DNA pellet in the buffer for the subsequent SalI digest?
Thank you for any suggestions!
Niko

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[Adam .]

I generally try to avoid purification of the DNA between digests, as you
lose a lot of it in the process. The way I've done sequential digest is to
cut with the enzyme which uses a low salt buffer, and then spike in salt to
adjust to the concentration of the second enzyme's buffer. Sometimes you
have to fudge a bit and use a non-optimal buffer for the first one.

An even simpler way is to use an isoschizomer for one of the enzymes so they
cut in the same buffer. In the past, I've had some finicky sequential
digests that worked beautifully if I just switched enzymes. I would highly
recommend doing this.

Adam

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[Michael Sullivan]

For the particular combination you are talking about, both the pH and
the salt concentrations of the buffers differ.

What I would probably do would be to digest first with Sac I in NEB
buffer 1. When you are satisfied the digest is complete (for example
by running an aliquot on a gel), I would then add 1/10 volume (that
is the final volume you expect with the added buffer and enzyme) NEB
buffer 3 and Sal I. For this combination of enzymes, this strategy
should work because both the buffer concentration and salt
concentration of NEB1 are very low and will have little impact on the
reaction conditions following addition of the NEB buffer 3.

If you do decide to purify following the first digest, don't waste
time prepping from a gel. In my mind, the only reason to gel purify
is if you only want one fragment from a digest that gives multiple
fragments. Precipitation is fine (and recovery is usually pretty good
unless you are starting with very small amounts of DNA). You can also
use a clean up type product (there are many)-- these are quick and
convenient.

Hope this helps.

Mike

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
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[Cathal Garvey]

I'm being forced to do likewise, with XhoI and SacI. It's a lot of
frustration and sequential purification (my method of choice, as Gel
Extractions are even less reliable) is my currently favoured method.

However, a quick look at the Fermentas FastDigest range of enzymes shows
that their speed (~15mins) is the least of their virtues; they all cut in
the same buffer, which incidentally contains loading dye for gel loading as
well.

They're expensive compared to the regular range, but I consider the extra
cost well worth it for the time saved and the satisfaction of a job done
quickly.

2009/11/19 Nikola Wenta <Nikola...@nottingham.ac.uk>

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[Nikola Wenta]

Dear all!
Of course, using isoschizomers or hifi enzymes are an interesting idea,
but are either time consuming or expensive for only one application. I
have therefore tried the low-salt-buffer-first suggestion, and it really
worked great! Complete digest, no waste of agarose and extraction kits,
simply two-step-single-tube ;-)
Thank you all very much for your suggestions!