Sequential restriction digest

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Thu Nov 19 17:35:21 EST 2009


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

On Nov 19, 2009, at 2:46 PM, Nikola Wenta 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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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
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