Sequential restriction digest

Adam . via methods%40net.bio.net (by anonwums1 from gmail.com)
Thu Nov 19 16:36:59 EST 2009


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

On Thu, Nov 19, 2009 at 2:46 PM, Nikola Wenta <Nikola.Wenta from nottingham.ac.uk
> wrote:

> 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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