Sequential restriction digest

[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.Wenta from nottingham.ac.uk>

> 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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