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 Digest, Multi-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 215 restriction enzymes are 100% active in CutSmart® Buffer, making double digestion simple. If you are using an enzyme that is not supplied with CutSmart Buffer, 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
Double digests with NEB's restriction enzymes can be set up in CutSmart 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: EcoRI, SspI 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 CutSmart 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, 1 X CutSmart and NEBuffer 1.1 do not contain any salt. 1X NEBuffer 2.1 contains 50 mM NaCl and 1X NEBuffer 3.1 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.
  1. DoubleDigestProtocol_video_thumb

    NEB Restriction Enzyme Double Digest Protocol

    Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.