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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 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
Enzy
me
AatII
AvrII
Bam
HI
BglII
BsgI
EagI
--
4
4
3
3
4
3
4
4
3
-4
seq
4
-3
seq
3
--
seq
2
3
4
4
3
seq
3
3
3
4
3
seq
4
seq
seq
2
4
3
EcoRI
3
3
3
EcoRI
-3
3
EcoRI
3
-seq
seq
EcoR 3
V
4
2
3
3
HindI 2
II
4
2
seq
1
4
NcoI 3
NdeI 4
NheI 2
NotI 3
PstI 3
PvuI 3
SacI 1
SacII 4
SalI 3
SmaI 4
SpeI 4
Sphl 2
XbaI 4
XhoI 4
XmaI 4
4
4
4
4
4
seq
4
seq
4
4
seq
4
4
4
4
4
4
1
4
4
4
2
3
3
2
1
4
3
4
4
2
4
4
4
seq
3
3
3
seq
3
3
3
seq
seq
3
seq
seq
3
3
3
seq
AvrII
Bam
HI
BglII
BsgI
EagI
NEBuff
er
EcoRI
EcoR
I
KpnI
MseI
MseI
NcoI
NdeI
NheI
NotI
PstI
PvuI
SacI
SacII
SalI
SmaI
SpeI
Sphl
XbaI
XhoI
XmaI
2
1
4
3
4
2
3
3
3
1
4
3
4
4
2
4
4
4
seq 4
EcoRI 2
EcoRI 3
4
2
seq
4
1
seq
4
4
3
4
4
3
4
4
3
4
2
seq
seq
3
3
4
3
3
seq
2
3
4
1
seq
4
4
seq
seq
3
3
4
4
seq
4
4
seq
4
2
3
4
4
3
4
4
3
4
4
seq
3
seq
-EcoRI
EcoRI
seq
EcoRI
--
2
2
seq
seq
2
seq
seq
1
2
4
3
EcoRI
3
4
3
EcoRI
3
4
3
EcoRI
2
4
seq
1
3
3
3
EcoRI
3
3
3
EcoRI
3
3
3
EcoRI
2
4
seq
1
2
4
seq
EcoRI
3
3
3
EcoRI
seq
4
seq
seq
2
4
seq
EcoRI
2
4
3
EcoRI
2
4
3
seq
3
4
3
EcoRI
2
4
seq
seq
4
3
EcoRI --
2
2
2
3
2
2
3
3
3
2
2
3
4
2
2
2
3
4
2
2
seq
seq
2
--
2
2
2
2
2
2
2
2
2
2
seq
4
2
2
2
2
seq
2
2
3
3
2
3
3
3
2
2
3
seq
2
2
2
3
2
seq
4
4
4
4
3
3
3
4
4
3
4
4
4
4
4
4
seq
3
3
3
seq
3
3
3
seq
seq
3
seq
seq
3
3
3
seq
1
EcoRI
EcoRI
EcoRI
1
EcoRI
EcoRI
EcoRI
1
EcoRI
EcoRI
seq
EcoRI
EcoRI
seq
EcoRI
seq
2
2
3
2
2
3
3
3
2
2
3
4
2
2
2
3
4
2
2
2
2
2
2
2
2
2
2
seq
4
2
2
2
2
seq
-1
1
1
1
2
1
2
1
4
seq
seq
1
1
2
1
4
1
-4
4
2
2
3
3
4
4
3
4
4
2
4
4
4
1
4
-4
2
3
3
3
1
4
3
4
4
2
4
4
4
1
4
4
-4
3
3
3
4
4
3
4
4
2
4
4
4
1
2
2
4
-2
2
2
1
4
seq
4
2
2
2
2
4
2
2
3
3
2
-3
3
2
2
3
seq
2
2
3
3
2
1
3
3
3
2
3
-3
1
2
3
4
2
2
3
3
4
2
3
3
3
2
3
3
-2
2
3
seq
2
2
3
3
2
1
4
1
4
1
2
1
2
-4
seq
4
1
1
4
1
4
4
4
4
4
4
2
2
2
4
-seq
4
4
4
4
4
4
seq
3
3
3
seq
3
3
3
seq
seq
-seq
seq
3
3
3
seq
seq
4
4
4
4
seq
4
seq
4
4
seq
-4
4
4
4
4
1
4
4
4
2
2
2
2
1
4
seq
4
-2
4
4
4
1
2
2
2
2
2
2
2
1
4
3
4
2
-2
2
4
2
4
4
4
2
3
3
3
4
4
3
4
4
2
-4
4
1
4
4
4
2
3
3
3
1
4
3
4
4
2
4
-4
4
4
4
4
4
2
4
2
4
4
seq
4
4
4
4
4
--
NEBuff
er
EcoRI
AatII
EcoR EcoR HindI
I
V
II
KpnI
3
3
4
3
EcoRI
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.
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.
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.
Double Digest reactions using standard restriction enzymes and RE-Mix are possible. There are a handful of enzymes which are not compatible with RE-Mix,
listed here. In this case, a sequential digest is recommended.
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 (NEB #B0101) (included in this chart and the Restriction
Enzyme Performance Chart), SspI (NEB #B0132) (same buffer composition as EcoRI) and DpnII (NEB #B0543). 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.
Double Digest FAQ Spotlight
Q:
I would like to digest DNA with EcoRI (NEB #R0101) and XbaI (NEB #R0145) 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 2.1. Why is a double digest not recommended with these
enzymes?
A: Although EcoRI shows 100% activity in NEBuffer 2.1, it also exhibits significant star activity in this buffer, resulting in non-specific, unintended cleavage. This is
also observed when using CutSmart Buffer. For this reason, a sequential digest is recommended. However, research has shown that EcoRI exhibits less star
activity in NEBuffers 1.1 and 3.1. Since XbaI has 75% activity in NEBuffer 3.1 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-Fidelity (HF) Format. HF Enzymes have been engineered for reduced star activity and have 100% activity in CutSmart Buffer
which will simplify your double digest.
HF™ is a trademark of New England Biolabs, Inc.
RE-Mix® is a registered trademark of New England Biolabs, Inc.