Download 窗体顶端 Select 1st enzyme: Select 2nd enzyme: 窗体底端 Enzyme

Select 1st
enzyme:
Enzyme Cat#
Select 2nd
enzyme:
Supplied
Temp
NEBuffer
GO
% Activity in
NEBuffer
Supplements
BSA
SAM
BamHI* R0136 37°C NEBuffer 3
Yes
No
75 100 100 100
KpnI*
Yes
No
100 75
R0142 37°C NEBuffer 1
1
2
3
0
4
50
Double Digest Recommendation(s) for BamHI + KpnI:

Double digest is not recommended. A sequential digest is required,
using each enzyme in its supplied NEBuffer.
Note: The above recommendation is based on the experimental results.
Please check Suggested NEBuffers for Double Digestion.
* BamHI has a High Fidelity version BamHI-HF (R3136)
KpnI has a High Fidelity version KpnI-HF (R3142)
High Fidelity (HF) Restriction Enzymes have been engineered for
reduced star activity
and have 100% activity in buffer 4 which may simplify your double
digest.
Double Digests
Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion)
provide reaction conditions that optimize enzyme activity as well as avoid star activity associate
supplied with its optimal NEBuffer to ensure 100% activity. NEBuffer compositions are listed on b
Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in t
Please use NEB Double Digest Finder to perform a double digest calculation.
Suggested NEBuffers for Double Digestion
Enzyme
AatIIAvrIIBamHIBglIIBsgI EagI EcoRIEcoRVHindIIIKpnI MseI NcoI NdeI N
NEBuffer
4
4
3
3
4
3
EcoRI
3
2
1
4
3
4
4
--
4
seq
seq
4
seq
seq
4
4
4
4
4
4
AvrII
4
4
--
3
2
4
3
EcoRI
2
2
1
4
4
4
BamHI
3
seq
3
--
3
3
3
EcoRI
3
seq
seq
3
3
3
BglII
3
seq
2
3
--
3
3
EcoRI
3
2
2
2
3
3
BsgI
4
4
4
3
3
--
seq
seq
4
2
seq
4
4
4
EagI
3
seq
3
3
3
seq
--
EcoRI
3
seq
seq
3
3
3
--
EcoRI
seq
1
AatII
EcoRI
EcoRI
seq EcoRI EcoRI EcoRI seq EcoRI
EcoRIEcoRIEcoRI
EcoRV
3
4
2
3
3
4
3
EcoRI
--
2
2
2
3
2
HindIII
2
4
2
seq
2
2
seq
seq
2
--
2
2
2
2
KpnI
1
4
1
seq
2
seq
seq
1
2
2
--
1
1
1
MseI
4
4
4
3
2
4
3
EcoRI
2
2
1
--
4
4
NcoI
3
4
4
3
3
4
3
EcoRI
3
2
1
4
--
4
NdeI
4
4
4
3
3
4
3
EcoRI
2
2
1
4
4
--
NheI
2
4
2
seq
2
4
seq
1
2
2
1
2
2
4
NotI
3
seq
3
3
3
3
3
EcoRI
3
2
2
2
3
3
PstI
3
4
3
3
3
3
3
EcoRI
3
2
1
3
3
3
PvuI
3
seq
2
3
3
3
3
EcoRI
3
2
2
3
3
3
SacI
1
4
1
seq
2
4
seq
1
2
2
1
4
1
4
SacII
4
4
4
seq
2
4
seq EcoRI
2
2
4
4
4
4
SalI
3
seq
3
3
3
3
3
EcoRI
3
seq
seq
3
3
3
SmaI
4
4
4
seq
seq
4
seq
seq
4
4
seq
4
4
4
SpeI
4
4
4
seq
2
4
seq EcoRI
2
2
1
4
4
4
SphI
2
4
2
3
2
4
3
EcoRI
2
2
1
2
2
2
XbaI
4
4
4
3
2
4
3
seq
2
2
2
4
4
4
XhoI
4
4
4
3
3
4
3
EcoRI
3
2
1
4
4
4
XmaI
4
4
4
seq
2
4
seq
seq
4
seq
4
4
4
4
Note: Enzymes in Dark Red are available in High Fidelity (HF) Format. HF Enzymes have been en
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
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 inh
enzyme).
Set up reaction according to recommended conditions. The final concentration of glycero
should be less than 5% to minimize the possibility of star activity. For example, in a 50 µl re
amount of enzyme added should not exceed 5 µl.
Incubate at recommended temperature. Overnight double digests should be avoide
possibility of star activity.


If two different incubation temperatures are necessary, choose the optimal rea
set up reaction accordingly. Add the first enzyme and incubate at the desired temperature,
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
may be adjusted to compensate for the slower rate of cleavage.
Setting up a Double Digestion with a Unique Buffer (designated “U”)

Our buffer system has been streamlined, leaving three enzymes that have unique buffers
in this chart and the Restriction Enzyme Activity Chart), SspI (same buffer composition as Eco
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 concentratio
recommended buffer and incubate to completion.
Adjust the salt concentration of the reaction (using a small volume of a concentrated sal
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 and XbaI at the same time. The Double Digest Finder
sequential digest, using each enzyme in its supplied NEBuffer. However, both of these enzym
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 th
in non-specific, unintended cleavage. This is also observed when using NEBuffer 4. For this reas
（相继的） digest is recommended. However, research has shown that EcoRI exhibits less st
NEBuffers 1 and 3. Since XbaI has 75% activity in NEBuffer 3 a double digest could be perform
provided that the reactions are set up according to the recommended reaction conditions for avo
In this case, EcoRI is available in High Fedelity (HF) Format. HF Enzymes have been engineere
activity and have 100% activity in NEBuffer 4 which may simplify your double digest.
Double Digest Finder
Cleaving a DNA substrate with two restriction enzymes simultaneously
(double digestion) is a common timesaving procedure. Use this tool to
select reaction conditions amenable to any two NEB restriction enzymes.
Click here for more information on double digests and a chart of double digests for some common
enzyme combinations.
Select 1st enzyme:
GO
Select 2nd enzyme:
Supplements
Enzyme
Cat#
Temp
BSA
SAM
1
2
3
4
BamHI-HF™
R3136
37°C
NEBuffer 4
No
No
100
50
10
100
KpnI-HF™
R3142
37°C
NEBuffer 4
No
No
100
25
0
100
Double Digest Recommendation(s) for BamHI-HF™ + KpnI-HF™:

% Activity in NEBuffer
Supplied NEBuffer
Digest in NEBuffer 4 at 37°C.