New England Biolabs provides a color-coded 10X NEBuffer with every restriction endonuclease to ensure 100% activity. 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. 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 and recommended diluent buffer.

Please click here for additional information about:
Unique NEBuffer Activity  | Double Digestion | Heat Inactivation | Activity at 37°C | Diluent Buffers | Time-Saver Enzymes

Please click here to request NEBuffer Activity Chart Poster

Click for the Chart Legend, Icon Description

Enzyme		Supplied NEBuffer	% Activity in NEBuffers				Heat Inactiva- tion °C	Incuba. Temp. °C	Diluent
			1	2	3	4
AatII		NEBuffer 4	0	50	50	100	65	37	A
Acc65I		NEBuffer 3 + BSA	10	75	100	25	65	37	A
AccI		NEBuffer 4	50	50	10	100	80	37	A
AciI		NEBuffer 3	25	50	100	50	65	37	A
AclI		NEBuffer 4 + BSA	10	10	0	100	No	37	B
AcuI		NEBuffer 2 + SAM	50	100	50	100	65	37	B
AfeI		NEBuffer 4	25	50	25	100	65	37	A
AflII		NEBuffer 2 + BSA	50	100	25	100	65	37	A
AflIII		NEBuffer 3 + BSA	25	75	100	50	80	37	B
AgeI		NEBuffer 1	100	50	10	75	65	37	C
AhdI		NEBuffer 4 + BSA	25	75	0	100	65	37	A
AleI		NEBuffer 4	10	25	10	100	65	37	A
AluI		NEBuffer 2	100	100	75	100	65	37	B
AlwI		NEBuffer 4	50	100	10	100	65	37	A
AlwNI		NEBuffer 4	50	100	50	100	65	37	A
ApaI		NEBuffer 4 + BSA	25	50	0	100	65	25	A
ApaLI		NEBuffer 4 + BSA	100	100	10	100	No	37	A
ApeKI		NEBuffer 3	25	75	100	50	No	75	B
ApoI		NEBuffer 3 + BSA	10	75	100	75	80	50	A
AscI		NEBuffer 4	0	10	10	100	65	37	A
AseI		NEBuffer 3	NR	75	100 dd	NR	65	37	B
AsiSI		NEBuffer 3 + BSA	50	100	100	50	80	37	B
AvaI		NEBuffer 4	10	75	10	100	80	37	A
AvaII		NEBuffer 4	50	75	10	100	65	37	A
AvrII		NEBuffer 2	100	100	50	100	No	37	B
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
BaeGI		NEBuffer 1	100	75	10	50	65	37	A
BaeI		NEBuffer 2 + BSA + SAM	50	100	50	75	65	25	A
BamHI		NEBuffer 3 + BSA	75	100	100 dd	100	No	37	A
BanI		NEBuffer 4	50	100	50	100	65	37	A
BanII		NEBuffer 4	100	10	50	100	65	37	A
BbsI		NEBuffer 2	100	100	25	75	65	37	B
BbvCI		NEBuffer 4	50	100	10	100	80	37	A
BbvI		NEBuffer 2	100	100	25	75	65	37	B
BccI		NEBuffer 1 + BSA	100	50	10	50	65	37	A
BceAI		NEBuffer 3 + BSA	100	100	100	100	65	37	A
BcgI		NEBuffer 3 + SAM	NR	NR	100 dd	NR	65	37	A
BciVI		NEBuffer 4	100	50	0	100	65	37	C
BclI		NEBuffer 3	50	100	100 dd	75	No	50	A
BfaI		NEBuffer 4	75	50	10	100	80	37	B
BfuAI		NEBuffer 3	0	75	100	10	65	50	B
BfuCI		NEBuffer 4 + BSA	100	50	25	100	80	37	B
BglI		NEBuffer 3	50	75	100	50	65	37	B
BglII		NEBuffer 3	10	75	100	10	No	37	A
BlpI		NEBuffer 4	50	100	10	100	No	37	A
BmgBI		NEBuffer 3 + BSA	0	25	100	10	65	37	C
BmrI		NEBuffer 2	75	100	75	100	65	37	B
BmtI		NEBuffer 2	25	100	25	50	65	37	B
BpmI		NEBuffer 3 + BSA	75	100	100	100	65	37	B
Bpu10I		NEBuffer 3	10	25	100	25	80	37	B
BpuEI		NEBuffer 2 + SAM	75	100	10	75	65	25	A
BsaAI		NEBuffer 3	75	75	100	75	80	37	A
BsaBI		NEBuffer 2	75	100	100	100	80	60	A
BsaHI		NEBuffer 4 + BSA	50	100	100	100	80	37	A
BsaI		NEBuffer 3	75	75	100	50	65	50	B
BsaJI		NEBuffer 2	100	100	100	100	80	60	A
BsaWI		NEBuffer 2 + BSA	50	100	50	50	80	60	A
BsaXI		NEBuffer 4	75	100	10	100	No	37	B
BseRI		NEBuffer 2	100	100	75	100	65	37	A
BseYI		NEBuffer 3	10	50	100	50	65	37	B
BsgI		NEBuffer 4 + SAM	50	75	50	100	65	37	B
BsiEI		NEBuffer 2 + BSA	50	100	10	100	80	60	A
BsiHKAI		NEBuffer 3 + BSA	50	100	100	100	80	65	B
BsiWI		NEBuffer 3	100	100	100	25	80	55	A
BslI		NEBuffer 3	10	50	100	75	80	55	A
BsmAI		NEBuffer 3	50	100	100	50	80	55	B
BsmBI		NEBuffer 3	75	100	100	100	80	55	B
BsmFI		NEBuffer 4 + BSA	25	50	50	100	80	65	A
BsmI		NEBuffer 2	75	100	75	75	80	65	A
BsoBI		NEBuffer 2	10	100	100	50	No	37	A
Bsp1286I		NEBuffer 4 + BSA	25	25	25	100	65	37	A
BspCNI		NEBuffer 1 + BSA + SAM	100	75	10	75	80	25	A
BspDI		NEBuffer 4	25	75	50	100	65	37	A
BspEI		NEBuffer 3	0	10	100	0	80	37	B
BspHI		NEBuffer 4	10	100	50	100	65	37	A
BspMI		NEBuffer 3	NR	NR	100	NR	65	37	B
BspQI		NEBuffer 3	10	50	100	100	80	50	B
BsrBI		NEBuffer 2	50	100	100	100	80	37	A
BsrDI		NEBuffer 2 + BSA	50	100	50	75	80	65	A
BsrFI		NEBuffer BsrFI	25	100	75	75	No	37	C
BsrGI		NEBuffer 2 + BSA	25	100	10	100	80	37	A
BsrI		NEBuffer 3	0	50	100	10	80	65	B
BssHII		NEBuffer 3	100	100	100 dd	100	80	50	A
BssKI		NEBuffer 3 + BSA	0	50	100	50	80	60	A
BssSI		NEBuffer 3	0	50	100 dd	10	80	37	B
BstAPI *		SEBuffer W	25	25	100	25	80	60	A
BstBI		NEBuffer 4	75	50	25	100	No	65	A
BstEII		NEBuffer 3 + BSA	50	75	100	75	No	60	A
BstNI		NEBuffer 2 + BSA	10	100	100	75	No	60	A
BstUI		NEBuffer 2	100	100	50	100	No	60	A
BstXI		NEBuffer 3	0	50	100	25	65	37	B
BstXI		NEBuffer 3	0	50	100	25	65	37	B
BstYI		NEBuffer 2	50	100	75	100	80	60	A
BstZ17I		NEBuffer 3	NR	NR	100	NR	No	37	B
Bsu36I		NEBuffer 3 + BSA	0	25	100	0	80	37	A
BtgI		NEBuffer 3 + BSA	25	50	100	100	80	37	A
BtgZI		NEBuffer 4 + BSA	10	25	0	100	80	60	A
BtsCI		NEBuffer 4	50	100	50	100	No	50	B
BtsI		NEBuffer 4 + BSA	100	50	50	100	80	55	A
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
Cac8I		NEBuffer 3	50	75	100	75	65	37	B
ClaI		NEBuffer 4 + BSA	10	50	50	100	65	37	A
CspCI		NEBuffer 2 + SAM	10	100	10	75	No	37	A
CviAII		NEBuffer 4	75	25	10	100	65	25	B
CviKI-1		NEBuffer 4	10	50	25	100	80	37	A
CviQI		NEBuffer 3 + BSA	75	100	100	75	No	25	B
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
DdeI		NEBuffer 3	75	100	100	75	65	37	A
DpnI		NEBuffer 4	100	100	75	100	80	37	B
DpnII		NEBuffer DpnII	NR	NR	100 dd	NR	65	37	B
DraI		NEBuffer 4	75	75	50	100	65	37	A
DraIII		NEBuffer 3 + BSA	100	75	100	25	65	37	B
DrdI		NEBuffer 4	25	50	10	100	65	37	A
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
EaeI		NEBuffer 1	100	100	50	100	65	37	A
EagI		NEBuffer 3	10	25	100	10	65	37	C
EarI		NEBuffer 1	100	100	50	100	65	37	A
EciI		NEBuffer 2 + BSA	50	100	100	50	65	37	A
EcoNI		NEBuffer 4	100	100	75	100	65	37	A
EcoO109I		NEBuffer 4 + BSA	100	100	75	100	65	37	A
EcoP15I		NEBuffer 3 + BSA + ATP	75	100	100	100	65	37	A
EcoRI		NEBuffer EcoRI dd	100	100	100	100	65	37	C
EcoRV		NEBuffer 3 + BSA	50	75	100	50	80	37	A
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
FatI		NEBuffer 2	10	100	50	50	65	55	A
FauI		NEBuffer 1	100	25	10	50	65	55	A
Fnu4HI		NEBuffer 4	10	25	25	100	65	37	A
FokI		NEBuffer 4	100	100	75	100	65	37	A
FseI		NEBuffer 4 + BSA	100	75	0	100	65	37	B
FspI		NEBuffer 4	10	75	10	100 dd	65	37	C
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
HaeII		NEBuffer 4 + BSA	75	100	50	100	80	37	A
HaeIII		NEBuffer 2	50	100	25	75	80	37	A
HgaI		NEBuffer 1	100	75	50	100	65	37	A
HhaI		NEBuffer 4 + BSA	75	100	100	100	65	37	A
HincII		NEBuffer 3 + BSA	75	100	100	100	65	37	B
HindIII		NEBuffer 2	50	100	10	50	65	37	B
HinfI		NEBuffer 2	75	100	75	75	80	37	A
HinP1I		NEBuffer 2	100	100	100	100	65	37	A
HpaI		NEBuffer 4	25	50	10	100	No	37	A
HpaII		NEBuffer 1	100	50	10	100	65	37	A
HphI		NEBuffer 4	NR	75	0	100	65	37	B
Hpy166II		NEBuffer 4	100	100	50	100	65	37	A
Hpy188I		NEBuffer 4	50	25	10	100	65	37	A
Hpy188III		NEBuffer 4 + BSA	100	100	10	100	65	37	A
Hpy99I		NEBuffer 4 + BSA	10	10	0	100	65	37	A
HpyAV		NEBuffer 4 + BSA	100	100	25	100	65	37	B
HpyCH4III		NEBuffer 4	100	50	25	100	80	37	A
HpyCH4IV		NEBuffer 1	100	25	10	25	65	37	A
HpyCH4V		NEBuffer 4	50	50	25	100	65	37	B
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
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	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
KasI		NEBuffer 2 + BSA	25	100	0	100	65	37	B
KpnI		NEBuffer 1 + BSA	100	75	0	50	No	37	A
Enzyme    top		Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
MboI		NEBuffer 3	75	100	100	100	65	37	A
MboII		NEBuffer 2	100	100	50	100	65	37	C
McrBC		NEBuffer 2 + BSA + GTP	50	100	10	75	65	37	B
MfeI		NEBuffer 4	75	50	10	100	65	37	A
MluI		NEBuffer 3	25	75	100	50	65	37	A
MlyI		NEBuffer 4 + BSA	50	50	25	100	65	37	A
MmeI		NEBuffer 4 + SAM	NR	NR	NR	100 dd	80	37	B
MnlI		NEBuffer 2 + BSA	75	100	50	75	65	37	B
MscI		NEBuffer 4	75	75	75	100	65	37	B
MseI