Nebuffer Performance Chart With Restriction Enzymes

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NEBuffer Performance Chart with Restriction Enzymes

For your convenience, New England Biolabs offers a simple 4 buffer system. A color-coded 10X NEBuffer is supplied with every restriction
endonuclease, ensuring 100% activity. Over 170 restriction enzymes exhibit 100% activity in NEBuffer 4, resulting in increased efficiency,
flexibility and ease of use especially when performing double digests.
To help select the best conditions for double digests, this chart shows the optimal (supplied) NEBuffer and approximate activity in the four
standard NEBuffers for each enzyme. In addition, the table shows recommended reaction temperature, heat inactivation temperature,
recommended diluent buffer and whether the enzyme is Time-Saver qualified (i.e., cleaves substrate in 5-15 minutes under recommended
conditions). Note: The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA.
Please check out other technical reference information related to restriction enzymes:
Double Digestion | Heat Inactivation | Activity at 37C | Diluent Buffers | Time-Saver Enzymes | High Fidelity (HF)
Restriction Enzymes
Chart Legend | Icon Dscriptions
Enzyme
Supplied NEBuffer
%Activity in NEBuffer
1
EcoRI
Heat Inac.
Incu. Temp.
Diluent
AatII
NEBuffer 4
50
50
100
25
65C
37C
Acc65I
NEBuffer 3 + BSA
10
75
100
25
50
65C
37C
AccI
NEBuffer 4
50
50
10
100
80C
37C
AciI
NEBuffer 3
25
50
100
50
100
65C
37C
AclI
NEBuffer 4 + BSA
10
10
100
37C
AcuI
NEBuffer 4 + SAM
50
100
50
100
100
65C
37C
AfeI
NEBuffer 4
25
50
25
100
100
65C
37C
AflII
NEBuffer 4 + BSA
50
100
25
100
65C
37C
AflIII
NEBuffer 3 + BSA
25
75
100
50
100
80C
37C
AgeI
NEBuffer 1
100
50
10
75
50
65C
37C
AgeI-HF
NEBuffer 4 + BSA
100
50
10
100
25
65C
37C
AgeI-HF RE-Mix
65C
37C
AhdI
NEBuffer 4 + BSA
25
75
100
10
65C
37C
AleI
NEBuffer 4
10
25
10
100
65C
37C
AluI
NEBuffer 4
100
100
75
100
100
65C
37C
AlwI
NEBuffer 4
50
100
10
100
65C
37C
AlwNI
NEBuffer 4
10
100
100
100
10
65C
37C
ApaI
NEBuffer 4 + BSA
25
50
100
65C
25C
ApaLI
NEBuffer 4 + BSA
100
100
10
100
37C
ApeKI
NEBuffer 3
25
75
100
50
100
75C
ApoI
NEBuffer 3 + BSA
10
75
100
75
100
80C
50C
AscI
NEBuffer 4
10
10
100
65C
37C
AscI RE-Mix
65C
37C
AseI
NEBuffer 3
N/R
75
100dd
N/R
50
65C
37C
AsiSI
NEBuffer 4 + BSA
50
100
100
100
80C
37C
AvaI
NEBuffer 4
10
75
10
100
50
80C
37C
AvaII
NEBuffer 4
50
75
10
100
50
65C
37C
AvrII
NEBuffer 4
100
100
50
100
50
80C
37C
BaeGI
NEBuffer 1
100
75
10
50
100
65C
37C
BaeI
NEBuffer 4 + BSA+SAM
50
100
50
100
40
65C
25C
BamHI
NEBuffer 3 + BSA
75
100
100dd
100
100
37C
BamHI-HF
NEBuffer 4
100
50
10
100
25
37C
BanI
NEBuffer 4 + BSA
50
100
50
100
25
65C
37C
BanII
NEBuffer 4
100
10
50
100
100
80C
37C
BbsI
NEBuffer 2
100
100
25
75
50
65C
37C
BbvCI
NEBuffer 4
50
100
10
100
100
80C
37C
BbvI
NEBuffer 2
100
100
25
75
100
65C
37C
BccI
NEBuffer 1 + BSA
100
50
10
50
15
65C
37C
BceAI
NEBuffer 3 + BSA
100
100
100
100
100
65C
37C
BcgI
NEBuffer 3 + SAM
N/R
N/R
100dd
N/R
50
65C
37C
BciVI
NEBuffer 4
100
50
100
25
65C
37C
BclI
NEBuffer 3
50
100
100dd
75
100
50C
BcoDI
NEBuffer 4
50
75
75
100
100
65C
37C
BfaI
NEBuffer 4
75
50
10
100
80C
37C
BfuAI
NEBuffer 3
75
100
10
100
65C
50C
BfuCI
NEBuffer 4 + BSA
100
50
25
100
15
80C
37C
BglI
NEBuffer 3
50
75
100
50
100
65C
37C
BglII
NEBuffer 3
10
75
100
10
100
37C
BlpI
NEBuffer 4
50
100
10
100
50
37C
BmgBI
NEBuffer 3 + BSA
25
100
10
50
65C
37C
BmrI
NEBuffer 2
75
100
75
100
50
65C
37C
BmtI
NEBuffer 2
25
100
25
50
100
65C
37C
BmtI-HF
NEBuffer 4 + BSA
50
100
10
100
N/R
65C
37C
BpmI
NEBuffer 3 + BSA
75
100
100
100
100
65C
37C
Bpu10I
NEBuffer 3
10
25
100
25
100
80C
37C
BpuEI
NEBuffer 4 + SAM
50
100
10
100
100
65C
37C
BsaAI
NEBuffer 4
100
100
100
100
100
37C
BsaBI
NEBuffer 4
50
100
75
100
100
80C
60C
BsaHI
NEBuffer 4 + BSA
50
100
100
100
100
80C
37C
BsaI
NEBuffer 4 + BSA
75
75
100
100
100
65C
37C
BsaI-HF
NEBuffer 4 + BSA
25
25
10
100
10
65C
37C
BsaJI
NEBuffer 4
100
100
100
100
100
80C
60C
BsaWI
NEBuffer 4 + BSA
50
100
50
100
40
80C
60C
BsaXI
NEBuffer 4
75
100
10
100
15
37C
BseRI
NEBuffer 4
100
100
75
100
100
65C
37C
BseYI
NEBuffer 3
10
50
100
50
100
65C
37C
BsgI
NEBuffer 4 + SAM
50
75
50
100
20
65C
37C
BsiEI
NEBuffer 4 + BSA
50
100
10
100
80C
60C
BsiHKAI
NEBuffer 4 + BSA
50
100
100
100
25
65C
BsiWI
NEBuffer 3
100
100
100
25
100
80C
55C
BslI
NEBuffer 3
10
50
100
75
100
80C
55C
BsmAI
NEBuffer 4
50
100
100
100
100
80C
55C
BsmBI
NEBuffer 3
75
100
100
100
100
80C
55C
BsmFI
NEBuffer 4 + BSA
25
50
50
100
15
80C
65C
BsmI
NEBuffer 4
75
100
75
100
80C
65C
BsoBI
NEBuffer 2
10
100
100
50
100
37C
Bsp1286I
NEBuffer 4 + BSA
25
25
25
100
10
65C
37C
BspCNI
NEBuffer 4 + BSA+SAM
100
75
10
100
15
80C
25C
BspDI
NEBuffer 4
25
75
50
100
100
65C
37C
BspEI
NEBuffer 3
10
100
N/R
80C
37C
BspHI
NEBuffer 4
10
100
50
100
65C
37C
BspMI
NEBuffer 3
N/R
N/R
100
N/R
100
65C
37C
BspQI
NEBuffer 4
10
50
100
100
100
80C
50C
BsrBI
NEBuffer 4
50
100
100
100
100
80C
37C
BsrDI
NEBuffer 2 + BSA
50
100
50
75
50
80C
65C
BsrFI
NEBuffer 4
10
100
100
100
100
37C
BsrGI
NEBuffer 2 + BSA
25
100
10
100
50
80C
37C
BsrI
NEBuffer 3
50
100
10
100
80C
65C
BssHII
NEBuffer 3
100
100
100dd
100
100
80C
50C
BssKI
NEBuffer 3 + BSA
50
100
50
100
80C
60C
BssSI
NEBuffer 3 + BSA
50
100dd
10
100
80C
37C
BstAPI
NEBuffer 4 + BSA
25
100
100
100
100
80C
60C
BstBI
NEBuffer 4
75
50
25
100
50
65C
BstEII
NEBuffer 3 + BSA
50
75
100
75
100
60C
BstEII-HF
NEBuffer 4 + BSA
50
75
100
100
N/R
37C
BstEII-HF RE-Mix
37C
BstNI
NEBuffer 2 + BSA
10
100
100
75
50
60C
BstUI
NEBuffer 4
100
100
50
100
100
60C
BstXI
NEBuffer 3
50
100
25
100
37C
65C
BstYI
NEBuffer 2
50
100
75
100
50
BstZ17I
NEBuffer 4
N/R
N/R
100
100
100
Bsu36I
NEBuffer 3 + BSA
25
100
15
BtgI
NEBuffer 3 + BSA
25
50
100
100
BtgZI
NEBuffer 4 + BSA
10
25
BtsCI
NEBuffer 4
50
100
BtsI
NEBuffer 4 + BSA
100
BtsIMutI
NEBuffer 4 + BSA
Cac8I
80C
60C
37C
80C
37C
100
80C
37C
100
50
80C
60C
50
100
25
50C
50
50
100
25
80C
55C
100
50
10
100
N/R
80C
55C
NEBuffer 4
50
75
100
100
50
65C
37C
ClaI
NEBuffer 4 + BSA
10
50
50
100
50
65C
37C
CspCI
NEBuffer 4 + SAM
10
100
10
100
20
65C
37C
CviAII
NEBuffer 4 + BSA
75
25
10
100
25
65C
25C
CviKI-1
NEBuffer 4
10
50
25
100
100
80C
37C
CviQI
NEBuffer 3 + BSA
75
100
100
75
25C
DdeI
NEBuffer 3
75
100
100
75
100
65C
37C
DpnI
NEBuffer 4
100
100
75
100
100
80C
37C
DpnII
NEBuffer DpnII
N/R
N/R
100dd
N/R
100
65C
37C
DraI
CutSmart Buffer
N/R
N/R
N/R
N/R
N/R
65C
37C
DraIII
NEBuffer 3 + BSA
100
75
100
25
50
65C
37C
DraIII-HF
NEBuffer 4 + BSA
50
10
100
50
37C
DrdI
NEBuffer 4 + BSA
25
50
10
100
100
65C
37C
EaeI
NEBuffer 4
100
100
50
100
100
80C
37C
EagI
NEBuffer 3
10
25
100
10
100
65C
37C
EagI-HF
NEBuffer 4
25
100
100
100
100
65C
37C
EarI
NEBuffer 4
100
100
50
100
100
65C
37C
EciI
NEBuffer 4
100
50
50
100
N/R
65C
37C
Eco53kI
NEBuffer 4
100
50
25
100
10
65C
37C
EcoNI
NEBuffer 4
100
100
75
100
100
65C
37C
EcoO109I
NEBuffer 4 + BSA
100
100
75
100
15
65C
37C
EcoP15I
NEBuffer 3 + BSA+ATP
75
100
100
100
50
65C
37C
EcoRI
NEBuffer EcoRI
100
100
100
100
100
65C
37C
EcoRI-HF
NEBuffer 4
10
100
100
65C
37C
EcoRI-HF RE-Mix
65C
37C
EcoRV
NEBuffer 3 + BSA
50
75
100
50
50
80C
37C
EcoRV-HF
NEBuffer 4
25
100
100
100
100
65C
37C
EcoRV-HF RE-Mix
65C
37C
FatI
NEBuffer 2
10
100
50
50
100
65C
55C
FauI
NEBuffer 4
100
50
10
100
50
65C
55C
Fnu4HI
NEBuffer 4
10
25
25
100
10
65C
37C
FokI
NEBuffer 4
100
100
75
100
25
65C
37C
FseI
NEBuffer 4 + BSA
100
75
100
50
65C
37C
FspEI
NEBuffer 4 + BSA
N/R
N/R
N/R
100
N/R
65C
37C
FspI
NEBuffer 4
10
75
10
100
50
65C
37C
HaeII
NEBuffer 4 + BSA
75
100
50
100
25
80C
37C
HaeIII
NEBuffer 4
50
100
25
100
100
80C
37C
HgaI
NEBuffer 1
100
75
50
100
25
65C
37C
HhaI
NEBuffer 4 + BSA
75
100
100
100
100
65C
37C
HincII
NEBuffer 3 + BSA
75
100
100
100
100
65C
37C
HindIII
NEBuffer 2
50
100
10
50
25
65C
37C
HindIII-HF
NEBuffer 4
10
100
10
100
10
80C
37C
HinfI
NEBuffer 4
75
100
75
100
100
80C
37C
HinP1I
NEBuffer 4
100
100
100
100
100
65C
37C
HpaI
NEBuffer 4
25
25
10
100
100
37C
HpaII
NEBuffer 1
100
50
10
50
15
65C
37C
HphI
NEBuffer 4
N/R
75
100
10
65C
37C
Hpy166II
NEBuffer 4
100
100
50
100
20
65C
37C
Hpy188I
NEBuffer 4
50
25
10
100
50
65C
37C
Hpy188III
NEBuffer 4 + BSA
100
100
10
100
65C
37C
Hpy99I
NEBuffer 4 + BSA
10
10
N/R
100
25
65C
37C
HpyAV
NEBuffer 4 + BSA
100
100
25
100
50
65C
37C
HpyCH4III
NEBuffer 4
100
50
25
100
20
80C
37C
HpyCH4IV
NEBuffer 1
100
25
10
25
15
65C
37C
HpyCH4V
NEBuffer 4
50
50
25
100
50
65C
37C
I-CeuI
NEBuffer 4 + BSA
10
10
100
N/R
65C
37C
I-SceI
NEBuffer I-SceI + BSA
10
50
50
50
N/R
65C
37C
KasI
NEBuffer 4 + BSA
25
100
100
100
65C
37C
KpnI
NEBuffer 1 + BSA
100
75
50
25
37C
KpnI-HF
NEBuffer 4
100
25
100
37C
KpnI-HF RE-Mix
37C
LpnPI
NEBuffer 4 + BSA
N/R
N/R
N/R
100
N/R
65C
37C
MboI
NEBuffer 4
75
100
100
100
100
65C
37C
MboII
NEBuffer 4
100
100
50
100
100
65C
37C
MfeI
NEBuffer 4
75
50
10
100
65C
37C
MfeI-HF
NEBuffer 4
75
50
10
100
65C
37C
MfeI-HF RE-Mix
65C
37C
MluCI
NEBuffer 4
100
10
10
100
80C
37C
MluI
NEBuffer 3
25
75
100
50
100
65C
37C
MlyI
NEBuffer 4 + BSA
50
50
25
100
25
65C
37C
MmeI
NEBuffer 4 + SAM
N/R
N/R
N/R
100dd
50
80C
37C
MnlI
NEBuffer 4 + BSA
75
100
50
100
50
65C
37C
MscI
NEBuffer 4
75
75
75
100
100
65C
37C
MseI
NEBuffer 4 + BSA
75
100
75
100
50
65C
37C
MslI
NEBuffer 4
100
100
25
100
10
65C
37C
MspA1I
NEBuffer 4 + BSA
50
100
50
100
50
65C
37C
MspI
NEBuffer 4
75
100
50
100
50
37C
MspJI
NEBuffer 4 + BSA
N/R
N/R
N/R
100
N/R
37C
MwoI
NEBuffer 3
10
75
100
75
100
60C
NaeI
NEBuffer 4
100
75
10
100
25
65C
37C
NarI
NEBuffer 4
100
75
75
100
50
65C
37C
Nb.BbvCI
NEBuffer 2
50
100
10
100
N/R
80C
37C
Nb.BsmI
NEBuffer 3
25
100
100
25
N/R
80C
65C
Nb.BsrDI
NEBuffer 2
10
100
25
50
N/R
80C
65C
Nb.BtsI
NEBuffer 4 + BSA
75
100
75
100
N/R
80C
37C
NciI
NEBuffer 4
100
25
10
100
25
37C
NcoI
NEBuffer 3
100
100
100
100
100
65C
37C
NcoI-HF
NEBuffer 4
50
100
10
100
100
80C
37C
NcoI-HF RE-Mix
80C
37C
NdeI
NEBuffer 4
75
100
75
100
100
65C
37C
NgoMIV
NEBuffer 4
100
50
10
100
80C
37C
NheI
NEBuffer 2 + BSA
100
100
10
100
15
65C
37C
NheI-HF
NEBuffer 4 + BSA
100
10
100
80C
37C
NheI-HF RE-Mix
80C
37C
NlaIII
NEBuffer 4 + BSA
25
25
25
100
65C
37C
NlaIV
NEBuffer 4 + BSA
10
10
10
100
100
65C
37C
NmeAIII
NEBuffer 4 + SAM
10
10
100
80C
37C
NotI
NEBuffer 3 + BSA
50
100
25
100
65C
37C
NotI-HF
NEBuffer 4 + BSA
25
100
25
100
50
65C
37C
NotI-HF RE-Mix
65C
37C
NruI
NEBuffer 3
10
100
10
100
65C
37C
NsiI
NEBuffer 3
10
75
100
25
100
80C
37C
NspI
NEBuffer 2 + BSA
100
100
100
65C
37C
Nt.AlwI
NEBuffer 2
25
100
25
50
N/R
80C
37C
Nt.BbvCI
NEBuffer 4
50
100
10
100
N/R
80C
37C
Nt.BsmAI
NEBuffer 4
100
50
10
100
N/R
65C
37C
Nt.BspQI
NEBuffer 3
10
75
100
25
N/R
80C
50C
65C
Nt.BstNBI
NEBuffer 3
10
100
10
N/R
80C
55C
Nt.CviPII
NEBuffer 4
25
100
50
100
N/R
65C
37C
PacI
NEBuffer 1 + BSA
100
75
10
100
65C
37C
PacI RE-Mix
65C
37C
PaeR7I
NEBuffer 4
25
100
10
100
50
37C
PciI
NEBuffer 3 + BSA
50
75
100
50
N/R
80C
37C
PflFI
NEBuffer 4 + BSA
25
25
100
65C
37C
PflMI
NEBuffer 3 + BSA
100
100
50
100
65C
37C
PhoI
NEBuffer 3
50
50
100
75
100
75C
PI-PspI
NEBuffer PI-PspI + BSA
10
10
10
N/R
65C
PI-SceI
NEBuffer PI-SceI + BSA
N/R
65C
37C
PleI
NEBuffer 4
10
100
100
100
50
65C
37C
PluTI
NEBuffer 4
100
25
100
N/R
65C
37C
PmeI
NEBuffer 4 + BSA
50
10
100
65C
37C
PmlI
NEBuffer 1 + BSA
100
75
75
15
65C
37C
PpuMI
NEBuffer 4
25
100
10
37C
PshAI
NEBuffer 4 + BSA
50
50
100
65C
37C
PsiI
NEBuffer 4
10
100
10
100
10
65C
37C
PspGI
NEBuffer 4
75
100
50
100
100
75C
PspOMI
NEBuffer 4
25
25
10
100
15
65C
37C
PspXI
NEBuffer 4
100
10
100
50
80C
37C
PstI
NEBuffer 3 + BSA
75
75
100
50
50
80C
37C
PstI-HF
NEBuffer 4
10
75
50
100
25
80C
37C
PvuI
NEBuffer 3 + BSA
10
75
100
10
100
37C
PvuI-HF
NEBuffer 4 + BSA
25
100
100
100
100
37C
PvuII
NEBuffer 2
100
100
100
100
100
37C
PvuII-HF
NEBuffer 4
25
100
80C
37C
RsaI
NEBuffer 4
100
100
50
100
15
65C
37C
RsrII
NEBuffer 4
25
75
10
100
25
65C
37C
SacI
NEBuffer 1 + BSA
100
50
10
100
15
65C
37C
SacI-HF
NEBuffer 4 + BSA
100
50
10
100
15
65C
37C
SacII
NEBuffer 4
25
75
10
100
100
65C
37C
SalI
NEBuffer 3 + BSA
100
50
65C
37C
SalI-HF
NEBuffer 4
10
100
100
100
100
65C
37C
SalI-HF RE-Mix
80C
37C
SapI
NEBuffer 4
75
50
100
50
65C
37C
Sau3AI
NEBuffer 1 + BSA
100
50
10
100
65C
37C
Sau96I
NEBuffer 4
50
100
100
100
50
80C
37C
SbfI
NEBuffer 4
75
50
100
25
65C
37C
SbfI-HF
NEBuffer 4
50
25
100
65C
37C
ScaI
NEBuffer 3
N/R
N/R
100dd
N/R
N/R
80C
37C
ScaI-HF
NEBuffer 4
100
100
10
100
15
65C
37C
ScaI-HF RE-Mix
65C
37C
ScrFI
NEBuffer 4
100
100
100
100
100
65C
37C
SexAI
NEBuffer 4 + BSA
100
75
50
100
50
65C
37C
SfaNI
NEBuffer 3
75
100
25
50
65C
37C
SfcI
NEBuffer 4 + BSA
75
50
10
100
25
65C
37C
SfiI
NEBuffer 4 + BSA
100
10
100
15
50C
SfoI
NEBuffer 4
25
100
50
100
100
37C
SgrAI
NEBuffer 4
100
50
10
100
50
65C
37C
SmaI
NEBuffer 4
100
N/R
65C
25C
SmlI
NEBuffer 4 + BSA
25
75
25
100
50
55C
SnaBI
NEBuffer 4 + BSA
25
50
25
100dd
80C
37C
SpeI
NEBuffer 4 + BSA
75
100
25
100
50
80C
37C
SpeI RE-Mix
80C
37C
SpeI-HF
NEBuffer 4 + BSA
25
50
100
N/R
80C
37C
SphI-HF
NEBuffer 4
50
25
10
100
65C
37C
SphI
NEBuffer 2
100
100
50
100
50
65C
37C
SspI
NEBuffer SspI
50
100
50
50
100
65C
37C
SspI-HF
NEBuffer 4
25
100
100
25
65C
37C
StuI
NEBuffer 4
100
100
50
100
50
65C
37C
StyD4I
NEBuffer 2
10
100
100
100
20
65C
37C
StyI
NEBuffer 3 + BSA
25
75
100
10
15
65C
37C
StyI-HF
NEBuffer 4
25
100
25
100
65C
37C
SwaI
NEBuffer 3 + BSA
10
10
100
10
15
65C
25C
TaqI
NEBuffer 4 + BSA
50
75
100
100
100
80C
65C
TfiI
NEBuffer 4
100
100
100
100
100
65C
TliI
NEBuffer 3 + BSA
25
25
100
10
50
75C
TseI
NEBuffer 4
75
100
100
100
100
65C
Tsp45I
NEBuffer 1 + BSA
100
25
75
50
65C
Tsp509I
NEBuffer 1
100
100
100
N/R
100
65C
TspMI
NEBuffer 4
50
75
50
100
20
75C
TspRI
NEBuffer 4 + BSA
25
50
25
100
65C
Tth111I
NEBuffer 4
50
25
25
100
100
65C
XbaI
NEBuffer 4 + BSA
100
75
100
15
65C
37C
XbaI RE-Mix
65C
37C
XcmI
NEBuffer 2
10
100
50
50
20
65C
37C
XhoI
NEBuffer 4 + BSA
75
100
100
100
100
65C
37C
XhoI RE-Mix
80C
37C
XmaI
NEBuffer 4 + BSA
25
50
100
25
65C
37C
XmnI
NEBuffer 4 + BSA
100
100
50
100
65C
37C
ZraI
NEBuffer 4
100
25
10
100
10
37C
Note:
1. SspI Unique Buffer has the same composition as EcoRI Unique Buffer.
2. The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA.

Nebuffer Performance Chart With Restriction Enzymes

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Nebuffer Performance Chart With Restriction Enzymes

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NEBuffer Performance Chart with Restriction Enzymes

NEBuffer I-SceI + BSA

NEBuffer PI-PspI + BSA

NEBuffer PI-SceI + BSA

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