Double Digest Protocol with Standard Restriction Enzymes also provides an interactive version of this protocol where you can discover and share optimizations with the research community. It is available for Single-temperature Double DigestMulti-temperature Double Digest (single buffer), and Sequential Double Digest

Digesting a DNA substrate with two restriction enzymes simultaneously (double digestion) is a common timesaving procedure. Over 210 restriction enzymes are 100% active in rCutSmart™ Buffer, making double digestion simple. If you are using an enzyme that is not supplied with rCutSmart the Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers.

Also, NEB's online tool NEBcloner® will help guide your reaction buffer selection when setting up double digests.

Setting up a Double Digestion
In most cases, double digests with NEB's restriction enzymes can be set up in rCutSmart Buffer. Otherwise, choose an NEBuffer that results in the most activity for both enzymes. If star activity is a concern, consider using one of our High Fidelity (HF®) enzymes.

Set up reaction according to recommended protocol. The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity. For example, in a 50 µl reaction, the total amount of enzyme added should not exceed 5 µl.

If two different incubation temperatures are necessary, choose the optimal reaction buffer and set up reaction accordingly. Add the first enzyme and incubate at the desired temperature. If the enzyme is heat inactivatable, a heat inactivation step is recommended. Add the second enzyme and incubate at the recommended temperature.

Setting up a Double Digestion with a Unique Buffer (designated “U”)
NEB currently supplies three enzymes with unique buffers: EcoRISspI and DpnII. In most cases, DpnII requires a sequential digest. Note that EcoRI and SspI have HF versions (EcoRI-HF and SspI-HF) which are supplied with rCutSmart Buffer. 

Setting up a Sequential Digestion
If there is no buffer in which the two enzymes exhibit > 50% activity, a sequential digest can be performed. Set up a reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer and incubate to completion. (For reference, 1X rCutSmart and NEBuffer r1.1 do not contain any salt. 1X NEBuffer r2.1 contains 50 mM NaCl and 1X NEBuffer r3.1 contains 100 mM NaCl.)

Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease.

Add the second enzyme and incubate to complete the second reaction.

Alternatively, a spin column (NEB #T1030) can be used to isolate the DNA prior to the second reaction.