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 and on the data card sent with each enzyme. The Activity
Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease
in the four standard NEBuffers and NEBuffer EcoRI. For some enzymes, a master mix format is available. Master Mixes can be used together in a double digest. See below for details.
Please use NEB Double Digest Finder to
perform a double digest calculation. |
Suggested NEBuffers for Double Digestion
 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Enzyme | | AatII | AvrII | BamHI | BglII | BsgI | EagI | EcoRI | EcoRV | HindIII | KpnI | MseI | NcoI | NdeI | NheI | NotI | PstI | PvuI | SacI | SacII | SalI | SmaI | SpeI | SphI | XbaI | XhoI | XmaI | | NEBuffer | 4 | 4 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 4 | 3 | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 4 | 2 | 4 | 4 | 4 | | AatII | 4 | -- | 4 | seq | seq | 4 | seq | seq | 4 | 4 | 4 | 4 | 4 | 4 | 4 | seq | 4 | seq | 4 | 4 | seq | 4 | 4 | 4 | 4 | 4 | 4 | | AvrII | 4 | 4 | -- | 3 | 2 | 4 | 3 | EcoRI | 2 | 2 | 1 | 4 | 4 | 4 | 2 | 3 | 3 | 2 | 1 | 4 | 3 | 4 | 4 | 2 | 4 | 4 | 4 | | BamHI | 3 | seq | 3 | -- | 3 | 3 | 3 | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | 3 | seq | seq | 3 | 3 | 3 | seq | | BglII | 3 | seq | 2 | 3 | -- | 3 | 3 | EcoRI | 3 | 2 | 2 | 2 | 3 | 3 | 2 | 3 | 3 | 3 | 2 | 2 | 3 | seq | 2 | 2 | 2 | 3 | 2 | | BsgI | 4 | 4 | 4 | 3 | 3 | -- | seq | seq | 4 | 2 | seq | 4 | 4 | 4 | 4 | 3 | 3 | 3 | 4 | 4 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | | EagI | 3 | seq | 3 | 3 | 3 | seq | -- | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | 3 | seq | seq | 3 | 3 | 3 | seq | | EcoRI | EcoRI | seq | EcoRI | EcoRI | EcoRI | seq | EcoRI | -- | EcoRI | seq | 1 | EcoRI | EcoRI | EcoRI | 1 | EcoRI | EcoRI | EcoRI | 1 | EcoRI | EcoRI | seq | EcoRI | EcoRI | seq | EcoRI | seq | | EcoRV | 3 | 4 | 2 | 3 | 3 | 4 | 3 | EcoRI | -- | 2 | 2 | 2 | 3 | 2 | 2 | 3 | 3 | 3 | 2 | 2 | 3 | 4 | 2 | 2 | 2 | 3 | 4 | | HindIII | 2 | 4 | 2 | seq | 2 | 2 | seq | seq | 2 | -- | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | seq | 4 | 2 | 2 | 2 | 2 | seq | | KpnI | 1 | 4 | 1 | seq | 2 | seq | seq | 1 | 2 | 2 | -- | 1 | 1 | 1 | 1 | 2 | 1 | 2 | 1 | 4 | seq | seq | 1 | 1 | 2 | 1 | 4 | | MseI | 4 | 4 | 4 | 3 | 2 | 4 | 3 | EcoRI | 2 | 2 | 1 | -- | 4 | 4 | 2 | 2 | 3 | 3 | 4 | 4 | 3 | 4 | 4 | 2 | 4 | 4 | 4 | | NcoI | 3 | 4 | 4 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 4 | -- | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 4 | 2 | 4 | 4 | 4 | | NdeI | 4 | 4 | 4 | 3 | 3 | 4 | 3 | EcoRI | 2 | 2 | 1 | 4 | 4 | -- | 4 | 3 | 3 | 3 | 4 | 4 | 3 | 4 | 4 | 2 | 4 | 4 | 4 | | NheI | 2 | 4 | 2 | seq | 2 | 4 | seq | 1 | 2 | 2 | 1 | 2 | 2 | 4 | -- | 2 | 2 | 2 | 1 | 4 | seq | 4 | 2 | 2 | 2 | 2 | 4 | | NotI | 3 | seq | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 2 | 2 | 3 | 3 | 2 | -- | 3 | 3 | 2 | 2 | 3 | seq | 2 | 2 | 3 | 3 | 2 | | PstI | 3 | 4 | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 1 | 3 | 3 | 3 | 2 | 3 | -- | 3 | 1 | 2 | 3 | 4 | 2 | 2 | 3 | 3 | 4 | | PvuI | 3 | seq | 2 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 2 | 3 | 3 | 3 | 2 | 3 | 3 | -- | 2 | 2 | 3 | seq | 2 | 2 | 3 | 3 | 2 | | SacI | 1 | 4 | 1 | seq | 2 | 4 | seq | 1 | 2 | 2 | 1 | 4 | 1 | 4 | 1 | 2 | 1 | 2 | -- | 4 | seq | 4 | 1 | 1 | 4 | 1 | 4 | | SacII | 4 | 4 | 4 | seq | 2 | 4 | seq | EcoRI | 2 | 2 | 4 | 4 | 4 | 4 | 4 | 2 | 2 | 2 | 4 | -- | seq | 4 | 4 | 4 | 4 | 4 | 4 | | SalI | 3 | seq | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | -- | seq | seq | 3 | 3 | 3 | seq | | SmaI | 4 | 4 | 4 | seq | seq | 4 | seq | seq | 4 | 4 | seq | 4 | 4 | 4 | 4 | seq | 4 | seq | 4 | 4 | seq | -- | 4 | 4 | 4 | 4 | 4 | | SpeI | 4 | 4 | 4 | seq | 2 | 4 | seq | EcoRI | 2 | 2 | 1 | 4 | 4 | 4 | 2 | 2 | 2 | 2 | 1 | 4 | seq | 4 | -- | 2 | 4 | 4 | 4 | | SphI | 2 | 4 | 2 | 3 | 2 | 4 | 3 | EcoRI | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 4 | 3 | 4 | 2 | -- | 2 | 2 | 4 | | XbaI | 4 | 4 | 4 | 3 | 2 | 4 | 3 | seq | 2 | 2 | 2 | 4 | 4 | 4 | 2 | 3 | 3 | 3 | 4 | 4 | 3 | 4 | 4 | 2 | -- | 4 | 4 | | XhoI | 4 | 4 | 4 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 4 | 4 | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 4 | 2 | 4 | -- | 4 | | XmaI | 4 | 4 | 4 | seq | 2 | 4 | seq | seq | 4 | seq | 4 | 4 | 4 | 4 | 4 | 2 | 4 | 2 | 4 | 4 | seq | 4 | 4 | 4 | 4 | 4 | -- |
Note: Enzymes in Dark Red are available in High Fidelity
(HF) Format. HF Enzymes have been engineered for reduced star activity and have 100% activity in NEBuffer
4 which may simplify your double digest. Click for more HF information.
Setting up a Double Digestion
- 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.
- If BSA is required for either enzyme, add it to the double digest reaction (BSA does not
inhibit any restriction enzyme).
- Set up reaction according to recommended conditions.
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.
- Incubate at recommended temperature. Overnight double digests should be avoided due to the
possibility of star activity.
- 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 kill 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 RE-Mix™ Master Mixes
- Set up reaction according to recommended conditions. Double Digest reactions using RE-Mix Master Mixes should be done in a 40 µl reaction volume, using 1 µg of DNA and 2 µl of each RE-Mix.
Setting up a Double Digestion with a Unique Buffer (designated “U”)
- Our buffer system has been streamlined, leaving three enzymes that have unique buffers:
EcoRI (included in this chart and the Restriction
Enzyme Activity Chart), SspI (same buffer composition as EcoRI) and DpnII. In most cases,
DpnII requires a sequential digest.
Setting up a Sequential Digestion
- 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.
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Double Digest FAQ Spotlight:
Q: I would like to digest DNA with EcoRI and XbaI at
the same time. The Double Digest Finder recommends a sequential digest, using each enzyme in its
supplied NEBuffer. However, both of these enzymes show 100% activity in NEBuffer 2. Why is a double
digest not recommended with these enzymes?
A: Although EcoRI shows 100% activity in NEBuffer 2, it also
exhibits significant star activity in this buffer, resulting in non-specific, unintended cleavage.
This is also observed when using NEBuffer 4. For this reason, a sequential digest is recommended.
However, research has shown that EcoRI exhibits less star activity in NEBuffers 1 and 3. Since XbaI
has 75% activity in NEBuffer 3 a double digest could be performed in this buffer, provided that the
reactions are set up according to the recommended reaction conditions for avoiding star
activity.
In this case, EcoRI is available in High Fedelity (HF) Format. HF Enzymes have been engineered for
reduced star activity and have 100% activity in NEBuffer 4 which may simplify your double digest. |
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