Double Digests

Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration. Each enzyme is supplied with its optimal NEBuffer to ensure 100% activity. NEBuffer compositions are listed on buffer pages. The Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers. NEB’s online tools, NEBcloner and Double Digest Finder 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 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. NEBcloner can also be used to determine recommended double digest conditions.
  • 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. Then, heat inactivate the first enzyme, add the second enzyme and incubate at the recommended temperature.
  • Depending on an enzyme's activity rating in a non-optimal NEBuffer, the number of units or incubation time may be adjusted to compensate for the slower rate of cleavage. The Performance Chart for Restriction Enzymes indicates if star activity is an issue with sub-optimal buffers.

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 has an HF version which is 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.
  • 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 can be used to isolate the DNA prior to the second reaction. NEB recommends using our Monarch nucleic acid purification kits.


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