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. On November 1, 2008, NEB changed the recommended buffer for 54 restriction enzymes to NEBuffer 4. With this change, NEB now provides over 160 restriction enzymes in a single buffer. This results 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 and NEBuffer EcoRI for each enzyme. If BSA is supplied with an enzyme, it is included in all NEBuffer activity reactions. In addition, the table also shows recommended reaction temperature, heat inactivation temperature, recommended diluent buffer and whether the enzyme is Time-Saver qualified (i.e., cleaves substrate in 5 minutes).

Please check out other technical reference information related to restriction enzymes:
Unique NEBuffer Activity  | Double Digestion | Heat Inactivation | Activity at 37°C | Diluent Buffers | Time-Saver Enzymes | High Fidelity (HF) Restriction Enzymes

Click for the Chart Legend, Icon Description

Enzyme		Supplied NEBuffer	% Activity in NEBuffers					Heat Inac. °C	Incu. Temp. °C	Dilu- ent
			1	2	3	4	EcoRI
AatII		NEBuffer 4	0	50	50	100	25	65	37	A
Acc65I		NEBuffer 3 + BSA	10	75	100	25	50	65	37	A
AccI		NEBuffer 4	50	50	10	100	5	80	37	A
AciI		NEBuffer 3	25	50	100	50	100	65	37	A
AclI		NEBuffer 4 + BSA	10	10	0	100	5	No	37	B
AcuI		NEBuffer 4 + SAM	50	100	50	100	100	65	37	B
AfeI		NEBuffer 4	25	50	25	100	100	65	37	A
AflII		NEBuffer 4 + BSA	50	100	25	100	0	65	37	A
AflIII		NEBuffer 3 + BSA	25	75	100	50	100	80	37	B
AgeI		NEBuffer 1	100	50	10	75	50	65	37	C
AhdI		NEBuffer 4 + BSA	25	75	0	100	10	65	37	A
AleI		NEBuffer 4	10	25	10	100	0	65	37	A
AluI		NEBuffer 4	100	100	75	100	100	65	37	B
AlwI		NEBuffer 4	50	100	10	100	5	65	37	A
AlwNI		NEBuffer 4	50	100	50	100	10	65	37	A
ApaI		NEBuffer 4 + BSA	25	50	0	100	5	65	25	A
ApaLI		NEBuffer 4 + BSA	100	100	10	100	5	No	37	A
ApeKI		NEBuffer 3	25	75	100	50	100	No	75	B
ApoI		NEBuffer 3 + BSA	10	75	100	75	100	80	50	A
AscI		NEBuffer 4	0	10	10	100	5	65	37	A
AseI		NEBuffer 3	NR	75	100 dd	NR	50	65	37	B
AsiSI		NEBuffer 4 + BSA	50	100	100	100	0	80	37	B
AvaI		NEBuffer 4	10	75	10	100	50	80	37	A
AvaII		NEBuffer 4	50	75	10	100	50	65	37	A
AvrII		NEBuffer 4	100	100	50	100	50	80	37	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
BaeGI		NEBuffer 1	100	75	10	50	100	65	37	A
BaeI		NEBuffer 4 + BSA + SAM	50	100	50	100	40	65	25	A
BamHI		NEBuffer 3 + BSA	75	100	100 dd	100	100	No	37	A
BamHI-HF™		NEBuffer 4	100	50	10	100	25	No	37	A
BanI		NEBuffer 4 + BSA	50	100	50	100	25	65	37	A
BanII		NEBuffer 4	100	10	50	100	100	80	37	A
BbsI		NEBuffer 2	100	100	25	75	50	65	37	B
BbvCI		NEBuffer 4	50	100	10	100	100	80	37	A
BbvI		NEBuffer 2	100	100	25	75	100	65	37	B
BccI		NEBuffer 1 + BSA	100	50	10	50	15	65	37	A
BceAI		NEBuffer 3 + BSA	100	100	100	100	100	65	37	A
BcgI		NEBuffer 3 + SAM	NR	NR	100 dd	NR	50	65	37	A
BciVI		NEBuffer 4	100	50	0	100	25	65	37	C
BclI		NEBuffer 3	50	100	100 dd	75	100	No	50	A
BfaI		NEBuffer 4	75	50	10	100	0	80	37	B
BfuAI		NEBuffer 3	0	75	100	10	100	65	50	B
BfuCI		NEBuffer 4 + BSA	100	50	25	100	15	80	37	B
BglI		NEBuffer 3	50	75	100	50	100	65	37	B
BglII		NEBuffer 3	10	75	100	10	100	No	37	A
BlpI		NEBuffer 4	50	100	10	100	50	No	37	A
BmgBI		NEBuffer 3 + BSA	0	25	100	10	50	65	37	C
BmrI		NEBuffer 2	75	100	75	100	50	65	37	B
BmtI		NEBuffer 2	25	100	25	50	100	65	37	B
BpmI		NEBuffer 3 + BSA	75	100	100	100	100	65	37	B
Bpu10I		NEBuffer 3	10	25	100	25	100	80	37	B
BpuEI		NEBuffer 4 + SAM	50	100	10	100	100	65	37	B
BsaAI		NEBuffer 4	100	100	100	100	100	No	37	C
BsaBI		NEBuffer 4	50	100	75	100	100	80	60	B
BsaHI		NEBuffer 4 + BSA	50	100	100	100	100	80	37	A
BsaI		NEBuffer 4	75	75	100	100	100	65	50	B
BsaJI		NEBuffer 4	100	100	100	100	100	80	60	A
BsaWI		NEBuffer 4 + BSA	50	100	50	100	40	80	60	A
BsaXI		NEBuffer 4	75	100	10	100	15	No	37	B
BseRI		NEBuffer 4	100	100	75	100	100	65	37	A
BseYI		NEBuffer 3	10	50	100	50	100	65	37	B
BsgI		NEBuffer 4 + SAM	50	75	50	100	20	65	37	B
BsiEI		NEBuffer 4 + BSA	50	100	10	100	5	80	60	A
BsiHKAI		NEBuffer 4 + BSA	50	100	100	100	25	No	65	B
BsiWI		NEBuffer 3	100	100	100	25	100	80	55	A
BslI		NEBuffer 3	10	50	100	75	-	80	55	A
BsmAI		NEBuffer 4	50	100	100	100	100	80	55	B
BsmBI		NEBuffer 3	75	100	100	100	100	80	55	B
BsmFI		NEBuffer 4 + BSA	25	50	50	100	15	80	65	A
BsmI		NEBuffer 4	75	100	75	100	5	80	65	A
BsoBI		NEBuffer 2	10	100	100	50	100	No	37	A
Bsp1286I		NEBuffer 4 + BSA	25	25	25	100	10	65	37	A
BspCNI		NEBuffer 4 + BSA + SAM	100	75	10	100	15	80	25	A
BspDI		NEBuffer 4	25	75	50	100	100	65	37	A
BspEI		NEBuffer 3	0	10	100	0	100	80	37	B
BspHI		NEBuffer 4	10	100	50	100	5	65	37	A
BspMI		NEBuffer 3	NR	NR	100	NR	100	65	37	B
BspQI		NEBuffer 4	10	50	100	100	100	80	50	B
BsrBI		NEBuffer 4	50	100	100	100	100	80	37	A
BsrDI		NEBuffer 2 + BSA	50	100	50	75	50	80	65	A
BsrFI		NEBuffer 4	10	100	100	100	100	No	37	C
BsrGI		NEBuffer 2 + BSA	25	100	10	100	50	80	37	A
BsrI		NEBuffer 3	0	50	100	10	100	80	65	B
BssHII		NEBuffer 3	100	100	100 dd	100	100	80	50	A
BssKI		NEBuffer 3 + BSA	0	50	100	50	100	80	60	A
BssSI		NEBuffer 3 + BSA	0	50	100 dd	10	100	80	37	B
BstAPI		NEBuffer 4 + BSA	25	100	100	100	100	80	60	A
BstBI		NEBuffer 4	75	50	25	100	50	No	65	A
BstEII		NEBuffer 3 + BSA	50	75	100	75	100	No	60	A
BstNI		NEBuffer 2 + BSA	10	100	100	75	50	No	60	A
BstUI		NEBuffer 4	100	100	50	100	100	No	60	A
BstXI		NEBuffer 3	0	50	100	25	100	65	37	B
BstYI		NEBuffer 2	50	100	75	100	50	80	60	A
BstZ17I		NEBuffer 4	NR	NR	100	100	100	No	37	B
Bsu36I		NEBuffer 3 + BSA	0	25	100	0	15	80	37	A
BtgI		NEBuffer 3 + BSA	25	50	100	100	100	80	37	B
BtgZI		NEBuffer 4 + BSA	10	25	0	100	50	80	60	A
BtsCI		NEBuffer 4	50	100	50	100	25	No	50	B
BtsI		NEBuffer 4 + BSA	100	50	50	100	25	80	55	A
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
Cac8I		NEBuffer 4	50	75	100	100	50	65	37	B
ClaI		NEBuffer 4 + BSA	10	50	50	100	50	65	37	A
CspCI		NEBuffer 4 + SAM	10	100	10	100	20	65	37	A
CviAII		NEBuffer 4	75	25	10	100	25	65	25	B
CviKI-1		NEBuffer 4	10	50	25	100	100	80	37	A
CviQI		NEBuffer 3 + BSA	75	100	100	75	0	No	25	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
DdeI		NEBuffer 3	75	100	100	75	100	65	37	A
DpnI		NEBuffer 4	100	100	75	100	100	80	37	B
DpnII		NEBuffer DpnII	NR	NR	100 dd	NR	100	65	37	B
DraI		NEBuffer 4	75	75	50	100	100	65	37	A
DraIII		NEBuffer 3 + BSA	100	75	100	25	50	65	37	B
DrdI		NEBuffer 4 + BSA	25	50	10	100	100	65	37	A
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
EaeI		NEBuffer 4	100	100	50	100	100	80	37	A
EagI		NEBuffer 3	10	25	100	10	100	65	37	C
EagI-HF™		NEBuffer 4	25	50	10	100	100	65	37	B
EarI		NEBuffer 4	100	100	50	100	100	65	37	A
EciI		NEBuffer 2 + BSA	50	100	100	50	100	65	37	A
Eco53kI		NEBuffer 4	100	50	25	100	-	65	37	A
EcoNI		NEBuffer 4	100	100	75	100	100	65	37	A
EcoO109I		NEBuffer 4 + BSA	100	100	75	100	15	65	37	A
EcoP15I		NEBuffer 3 + BSA + ATP	75	100	100	100	50	65	37	A
EcoRI		NEBuffer EcoRI dd	100	100	100	100	100	65	37	C
EcoRI-HF™		NEBuffer 4	10	100	0	100	5	65	37	C
EcoRV		NEBuffer 3 + BSA	50	75	100	50	50	80	37	A
EcoRV-HF™		NEBuffer 4	25	100	100	100	100	65	37	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
FatI		NEBuffer 2	10	100	50	50	100	65	55	A
FauI		NEBuffer 4	100	50	10	100	50	65	55	A
Fnu4HI		NEBuffer 4	10	25	25	100	10	65	37	A
FokI		NEBuffer 4	100	100	75	100	25	65	37	A
FseI		NEBuffer 4 + BSA	100	75	0	100	50	65	37	B
FspI		NEBuffer 4	10	75	10	100 dd	50	65	37	C
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
HaeII		NEBuffer 4 + BSA	75	100	50	100	25	80	37	A
HaeIII		NEBuffer 4	50	100	25	100	100	80	37	A
HgaI		NEBuffer 1	100	75	50	100	25	65	37	A
HhaI		NEBuffer 4 + BSA	75	100	100	100	100	65	37	A
HincII		NEBuffer 3 + BSA	75	100	100	100	100	65	37	B
HindIII		NEBuffer 2	50	100	10	50	25	65	37	B
HinfI		NEBuffer 4	75	100	75	100	100	80	37	A
HinP1I		NEBuffer 4	100	100	100	100	100	65	37	A
HpaI		NEBuffer 4	25	50	10	100	100	No	37	A
HpaII		NEBuffer 1	100	50	10	50	15	65	37	A
HphI		NEBuffer 4	NR	75	0	100	10	65	37	B
Hpy166II		NEBuffer 4	100	100	50	100	20	65	37	C
Hpy188I		NEBuffer 4	50	25	10	100	50	65	37	A
Hpy188III		NEBuffer 4 + BSA	100	100	10	100	5	65	37	A
Hpy99I		NEBuffer 4 + BSA	10	10	0	100	25	65	37	A
HpyAV		NEBuffer 4 + BSA	100	100	25	100	50	65	37	B
HpyCH4III		NEBuffer 4	100	50	25	100	20	80	37	A
HpyCH4IV		NEBuffer 1	100	25	10	25	15	65	37	A
HpyCH4V		NEBuffer 4	50	50	25	100	50	65	37	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
I-CeuI		NEBuffer 4 + BSA	10	10	0	100	-	65	37	B
I-SceI		NEBuffer I-SceI + BSA	10	50	50	50	-	65	37	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
KasI		NEBuffer 4 + BSA	25	100	0	100	100	65	37	B
KpnI		NEBuffer 1 + BSA	100	75	0	50	25	No	37	A
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
MboI		NEBuffer 4	75	100	100	100	100	65	37	A
MboII		NEBuffer 4	100	100	50	100	100	65	37	C
MfeI		NEBuffer 4	75	50	10	100	5	65	37	A
MfeI-HF™		NEBuffer 4	75	50	10	100	0	65	37	A
MluI		NEBuffer 3	25	75	100	50	100	65	37	A
MlyI		NEBuffer 4 + BSA	50	50	25	100	25	65	37	A
MmeI		NEBuffer 4 + SAM	NR	NR	NR	100 dd	50	80	37	B
MnlI		NEBuffer 4 + BSA	75	100	50	100	50	65	37	B
MscI		NEBuffer 4	75	75	75	100	100	65	37	B
MseI		NEBuffer 4 + BSA	75	100	75	100	50	65	37	A
MslI		NEBuffer 4	100	100	25	100	10	65	37	A
MspA1I		NEBuffer 4 + BSA	50	100	50	100	50	65	37	B
MspI		NEBuffer 4	75	100	50	100	50	80	37	A
MwoI		NEBuffer 3	10	75	100	75	100	No	60	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
NaeI		NEBuffer 4	100	75	10	100	25	65	37	A
NarI		NEBuffer 4	100	75	75	100	50	65	37	A
Nb.BbvCI		NEBuffer 2	50	100	10	100	-	80	37	A
Nb.BsmI		NEBuffer 3	25	100	100	25	-	80	65	A
Nb.BsrDI		NEBuffer 2	10	100	25	50	-	80	65	A
Nb.BtsI		NEBuffer 4 + BSA	75	100	75	100	-	80	37	A
NciI		NEBuffer 4	100	25	10	100	25	No	37	A
NcoI		NEBuffer 3	100	100	100	100	100	65	37	A
NcoI-HF™		NEBuffer 4	50	100	10	100	100	80	37	B
NdeI		NEBuffer 4	75	100	75	100	100	65	37	A
NgoMIV		NEBuffer 4	100	50	10	100	0	80	37	A
NheI		NEBuffer 2 + BSA	100	100	10	100	15	65	37	C
NheI-HF™		NEBuffer 4 + BSA	100	10	0	100	0	80	37	C
NlaIII		NEBuffer 4 + BSA	25	25	25	100	5	65	37	B
NlaIV		NEBuffer 4 + BSA	10	10	10	100	100	65	37	B
NmeAIII		NEBuffer 4 + SAM	10	10	0	100	0	80	37	B
NotI		NEBuffer 3 + BSA	0	50	100	25	100	65	37	C
NotI-HF™		NEBuffer 4 + BSA	25	100	25	100	50	65	37	A
NruI		NEBuffer 3	0	10	100	10	100	65	37	A
NsiI		NEBuffer 3	10	75	100	25	100	80	37	A
NspI		NEBuffer 2 + BSA	100	100	0	100	5	65	37	A
Nt.AlwI		NEBuffer 2	25	100	25	50	-	80	37	A
Nt.BbvCI		NEBuffer 4	50	100	10	100	-	80	37	A
Nt.BsmAI		NEBuffer 4	100	50	10	100	-	65	37	A
Nt.BspQI		NEBuffer 3	10	75	100	25	-	80	50	B
Nt.BstNBI		NEBuffer 3	0	10	100	10	-	80	55	A
Nt.CviPII		NEBuffer 4	25	100	50	100	-	65	37	A
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
PacI		NEBuffer 1 + BSA	100	75	10	100	5	65	37	A
PaeR7I		NEBuffer 4	25	100	10	100	50	No	37	A
PciI		NEBuffer 3	50	75	100	50	0	80	37	A
PflFI		NEBuffer 4 + BSA	0	25	25	100	-	65	37	A
PflMI		NEBuffer 3 + BSA	0	100	100	50	100	65	37	A
PhoI		NEBuffer 3	50	50	100	75	100	No	75	A
PI-PspI		NEBuffer PI-PspI + BSA	0	10	10	10	-	No	65	B
PI-SceI		NEBuffer PI-SceI + BSA	0	0	0	0	-	65	37	B
PleI		NEBuffer 4	100	100	75	100	50	65	37	A
PmeI		NEBuffer 4 + BSA	0	50	10	100	5	65	37	A
PmlI		NEBuffer 1 + BSA	100	75	0	75	15	65	37	A
PpuMI		NEBuffer 4	0	25	0	100	10	No	37	B
PshAI		NEBuffer 4 + BSA	50	75	10	100	5	65	37	A
PsiI		NEBuffer 4	10	100	10	100	10	65	37	B
PspGI		NEBuffer 4	75	100	50	100	100	No	75	A
PspOMI		NEBuffer 4	25	25	10	100	15	65	37	A
PspXI		NEBuffer 4	0	100	10	100	50	80	37	B
PstI		NEBuffer 3 + BSA	75	75	100	50	50	80	37	C
PvuI		NEBuffer 3 + BSA	10	75	100	10	100	80	37	B
PvuII		NEBuffer 2	100	100	100	100	100	No	37	B
PvuII-HF™		NEBuffer 4	0	25	0	100	0	80	37	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
RsaI		NEBuffer 4	100	100	50	100	15	65	37	A
RsrII		NEBuffer 4	25	75	10	100	25	65	37	C
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
SacI		NEBuffer 1 + BSA	100	50	10	100	15	65	37	A
SacI-HF™		NEBuffer 4 + BSA	100	50	10	100	15	65	37	A
SacII		NEBuffer 4	25	75	10	100	100	65	37	A
SalI		NEBuffer 3 + BSA	0	0	100	0	50	65	37	A
SalI-HF™		NEBuffer 4	10	100	100	100	100	65	37	A
SapI		NEBuffer 4	75	50	0	100	50	65	37	C
Sau3AI		NEBuffer 1 + BSA	100	50	10	100	5	65	37	A
Sau96I		NEBuffer 4	50	100	100	100	50	80	37	A
SbfI		NEBuffer 4	75	50	0	100	25	65	37	A
SbfI-HF™		NEBuffer 4	50	25	0	100	0	65	37	B
ScaI		NEBuffer 3	NR	NR	100 dd	NR	50	80	37	A
ScaI-HF™		NEBuffer 4	100	100	10	100	15	65	37	B
ScrFI		NEBuffer 4	100	100	100	100	100	65	37	A
SexAI		NEBuffer 4 + BSA	100	75	50	100	50	65	37	A
SfaNI		NEBuffer 3	0	75	100	25	50	65	37	B
SfcI		NEBuffer 4 + BSA	75	50	10	100	25	65	37	B
SfiI		NEBuffer 4 + BSA	0	100	10	100	15	No	50	C
SfoI		NEBuffer 4	25	100	50	100	100	No	37	B
SgrAI		NEBuffer 4	100	50	10	100	50	65	37	A
SmaI		NEBuffer 4	0	0	0	100	0	65	25	A
SmlI		NEBuffer 4 + BSA	25	75	25	100	50	No	55	A
SnaBI		NEBuffer 4 + BSA	25	50	25	100 dd	5	80	37	A
SpeI		NEBuffer 4 + BSA	75	100	25	100	50	80	37	C
SphI		NEBuffer 2	100	100	50	100	50	65	37	B
SphI-HF™		NEBuffer 4	50	25	10	100	0	65	37	B
SspI		NEBuffer SspI	50	100	50	50	100	65	37	C
SspI-HF™		NEBuffer 4	25	100	0	100	25	65	37	B
StuI		NEBuffer 4	100	100	50	100	50	65	37	A
StyD4I		NEBuffer 2	10	100	100	100	20	65	37	B
StyI		NEBuffer 3 + BSA	25	75	100	10	15	65	37	A
SwaI		NEBuffer 3 + BSA	10	10	100	10	15	65	25	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
TaqαI		NEBuffer 4 + BSA	50	75	100	100	-	80	65	B
TfiI		NEBuffer 4	100	100	100	100	100	No	65	C
TliI		NEBuffer 3 + BSA	25	25	100	10	50	No	75	B
TseI		NEBuffer 4	75	100	100	100	100	No	65	B
Tsp45I		NEBuffer 1 + BSA	100	25	0	75	50	No	65	A
Tsp509I		NEBuffer 1	100	100	100	NR	100	No	65	A
TspMI		NEBuffer 4	50	75	50	100	20	No	75	B
TspRI		NEBuffer 4 + BSA	25	50	25	100	5	No	65	B
Tth111I		NEBuffer 4	50	25	25	100	100	No	65	B
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
XbaI		NEBuffer 4 + BSA	0	100	75	100	15	65	37	A
XcmI		NEBuffer 2	10	100	50	50	20	65	37	C
XhoI		NEBuffer 4 + BSA	75	100	100	100	100	65	37	A
XmaI		NEBuffer 4 + BSA	25	50	0	100	25	65	37	A
XmnI		NEBuffer 4 + BSA	100	100	50	100	5	65	37	A
Enzyme    top		Supplied NEBuffer	B1	B2	B3	B4	EcoRI	Heat Inac.	Incu. Temp.	Dilu- ent
ZraI		NEBuffer 4	100	25	10	100	10	No	37	A

Note: The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA.