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. For some enzymes, a master mix format is available. Master Mixes can be used together in a double digest. See below for details.

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. 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.

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.

Setting up a Double Digestion with RE-Mix® Master Mixes

RE-Mix master mixes can also be used in double digest reactions with another RE-Mix or with standard restriction enzymes with 37°C incubation temperatures.

  RE-Mix Standard Restriction Enzymes
DNA   X μl (up to 1 μg)  X μl (up to 1 μg)
 dH2O  36 μl–X  17 μl–X
 RE-Mix 1  2 μl  2 μl
 RE-Mix 2 or Standard Restriction Enzyme  2 μl  1 μl
 Total volume  40 μl  20 μl
 Incubation Temperature  37°C  37°C
 Incubation Time  15 minutes  15 minutes*

* If standard restriction enzyme is Time-Saver™ qualified; 1 hour if not Time-Saver qualified.

Some Standard Restriction Enzymes are not compatible with RE-Mix

Standard Restriction Enzymes Not Compatible with RE-Mix Master Mixes
(Sequential Digestions Recommended)
AleI DpnII SalI
AlwI MluI SexAI
BanI MwoI SfaNI
BfaI NotI SphI-HF
BspCNI PacI StyI
BspEI PvuI StyD4I
BtgZI RsrII Tsp45I

CutSmart® and HF® are trademarks of New England Biolabs, Inc.
RE-Mix® is a registered trademark of New England Biolabs, Inc.

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