NEBuffer Chart

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. Incubation temperature is 37°C unless otherwise noted. If BSA is supplied with an enzyme, it is included in all NEBuffer activity reactions. Additional double digestion information can be found here.

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

Buffers should be thawed completely and mixed thoroughly before using.

Unique NEBuffer Chart

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| A | B | C | D | E | F | H | K | M | N | P | R | S | T | X | Z |

Enzyme	Supplied NEBuffer	% Activity in NEBuffers
Enzyme	Supplied NEBuffer	1	2	3	4
AatII	4	0	50	50	100
AccI	4	50	50	10	100
Acc65I	3 + BSA	10	75	100	25
AciI	3	25	50	100	50
AclI	4 + BSA	10	10	0	100
AcuI	2 + SAM	50	100	50	100
AfeI	4	25	50	25	100
AflII	2 + BSA	50	100	25	100
AflIII	3 + BSA	25	75	100	50
AgeI	1	100	50	10	75
AhdI	4 + BSA	25	75	0	100
AleI	4	10	25	10	100
AluI	2	100	100	75	100
AlwI	4	50	100	10	100
AlwNI	4	50	100	50	100
ApaI @25°C	4 + BSA	25	50	0	100
ApaLI	4 + BSA	100	100	10	100
ApeKI @75°C	3	25	75	100	50
ApoI @50°C	3 + BSA	10	75	100	75
AscI	4	0	10	10	100
AseI	3	NR	75	100^dd	NR
AsiSI	3 + BSA	50	100	100	50
AvaI	4	10	75	10	100
AvaII	4	50	75	10	100
AvrII	2	100	100	50	100
BaeI @25°C	2 + BSA + SAM	50	100	50	75
BamHI	3 + BSA	75	100	100^dd	100
BanI	4	50	100	50	100
BanII	4	100	10	50	100
BbsI	2	100	100	25	75
BbvI	2	100	100	25	75
BbvCI	4	50	100	10	100
BccI	1 + BSA	100	50	10	50
BceAI	3 + BSA	100	100	100	100
BcgI	3 + SAM	NR	NR	100^dd	NR
BciVI	4	100	25	10	100
BclI @50°C	3	50	100	100^dd	75
BfaI	4	75	50	10	100
BfuAI @50°C	3	0	75	100	10
BfuCI	4 + BSA	100	50	25	100
BglI	3	50	75	100	50
BglII	3	10	75	100	10
BlpI	4	50	100	10	100
Bme1580I	2	75	100	75	75
BmgBI	3 + BSA	0	25	100	10
BmrI	2	75	100	75	100
BmtI	2	25	100	25	50
BpmI	3 + BSA	75	100	100	100
Bpu10I	3	10	25	100	25
BpuEI @25°C	2 + SAM	75	100	10	75
BsaI @50°C	3	75	75	100	100
BsaAI	3	75	75	100	75
BsaBI @60°C	2	75	100	100	100
BsaHI	4 + BSA	50	100	100	100
BsaJI @60°C	2	100	100	100	100
BsaWI @60°C	2 + BSA	50	100	50	50
BsaXI	4	75	100	10	100
BseRI	2	100	100	75	100
BseYI	3	10	50	100	50
BsgI	4 + SAM	50	75	50	100
BsiEI @60°C	2 + BSA	50	100	10	100
BsiHKAI @65°C	3 + BSA	50	100	100	100
BsiWI @55°C	3	100	100	100	25
BslI @55°C	3	10	50	100	75
BsmI @65°C	2	75	100	75	75
BsmAI @55°C	3	50	100	100	50
BsmBI @55°C	3	75	100	100	100
BsmFI @65°C	4 + BSA	25	50	50	100
BsoBI	2	10	100	100	50
Bsp1286I	4 + BSA	25	25	25	100
BspCNI @25°C	1 + BSA + SAM	100	75	10	75
BspDI	4	25	75	50	100
BspEI	3	0	10	100	0
BspHI	4	10	100	50	100
BspMI	3	NR	NR	100	NR
BspQI	3	10	50	100	100
BsrI @65°C	3	0	50	100	10
BsrBI	2	50	100	100	100
BsrDI @65°C	2 + BSA	50	100	50	75
BsrFI	U	25	100	75	75
BsrGI	2 + BSA	25	100	10	100
BssHII @50°C	3	100	100	100^dd	100
BssKI @60°C	3 + BSA	0	50	100	50
BssSI	3	0	50	100^dd	10
BstAPI* @60°C	SE-W	25	25	100	25
BstBI @65°C	4	75	50	25	100
BstEII @60°C	3 + BSA	50	75	100	75
BstNI @60°C	2 + BSA	10	100	100	75
BstUI @60°C	2	100	100	50	100
BstXI @55°C	3	25	100	100	50
BstYI @60°C	2	50	100	75	100
BstZ17I	3	NR	NR	100	NR
Bsu36I	3 + BSA	0	25	100	0
BtgI	3 + BSA	25	50	100	100
BtgZI @60°C	4 + BSA	10	25	0	100
BtsI	4 + BSA	100	50	50	100
BtsCI	4	50	100	50	100
Cac8 I	3	50	75	100	75
ClaI	4 + BSA	10	50	50	100
CspCI	2 + SAM	10	100	10	75
CviAII @25°C	4	75	25	10	100
CviKI-1	4	10	50	25	100
CviQI	3 + BSA	75	100	100	75
DdeI	3	75	100	100	75
DpnI	4	100	100	75	100
DpnII	U	NR	NR	100^dd	NR
DraI	4	75	75	50	100
DraIII	3 + BSA	100	75	100	25
DrdI	4	25	50	10	100
EaeI	1	100	100	50	100
EagI	3	10	25	100	10
EarI	1	100	100	50	100
EciI	2 + BSA	50	100	100	50
EcoNI	4	100	100	75	100
EcoO109I	4 + BSA	100	100	75	100
EcoP15I	3 + BSA + ATP	75	100	100	100
EcoRI	U^dd	100	100	100	100
EcoRV	3 + BSA	50	75	100	50
FatI @55°C	2	10	100	50	50
FauI @55°C	1	100	25	10	50
Fnu4HI	4	10	25	25	100
FokI	4	100	100	75	100
FseI	4 + BSA	100	75	0	100
FspI	4	10	75	10	100
HaeII	4 + BSA	75	100	50	100
HaeIII	2	50	100	25	75
HgaI	1	100	75	50	100
HhaI	4 + BSA	75	100	100	100
HincII	3 + BSA	75	100	100	100
HindIII	2	50	100	10	50
HinfI	2	75	100	75	75
HinP1I	2	100	100	100	100
HpaI	4	25	50	10	100
HpaII	1	100	50	10	100
HphI	4	NR	75	0	100
Hpy166II	4	100	100	50	100
Hpy188I	4	50	25	10	100
Hpy188III	4 + BSA	100	100	10	100
Hpy99I	4 + BSA	10	10	0	100
HpyAV	4 + BSA	100	100	25	100
HpyCH4III	4	100	50	25	100
HpyCH4IV	1	100	25	10	25
HpyCH4V	4	50	50	25	100
I-CeuI	4 + BSA	10	10	0	100
I-SceI	U + BSA	10	50	50	50
KasI	2 + BSA	75	100	75	75
KpnI	1 + BSA	100	75	0	50
MboI	3	75	100	100	100
MboII	2	100	100	50	100
McrBC	2 + BSA + GTP	50	100	10	75
MfeI	4	75	50	10	100
MluI	3	25	75	100	50
MlyI	4 + BSA	50	50	25	100
MmeI	4 + SAM	NR	NR	NR	^dd100
MnlI	2 + BSA	75	100	50	75
MscI	4	75	75	75	100
MseI	2 + BSA	75	100	75	75
MslI	2	100	100	25	100
MspI	2	75	100	50	50
MspA1I	4 + BSA	50	100	50	100
MwoI @60°C	3	10	75	100	75
N.AlwI	2	25	100	25	50
Nb.BbvCI	2	50	100	10	100
Nb.BsrDI @65°C	2	10	100	25	50
Nb.BtsI	4 + BSA	75	100	75	100
Nt.BbvCI	4	50	100	10	100
Nt.BspQI	3	10	75	100	25
Nt.BstNBI @55°C	3	0	10	100	10
Nt.CviPII	4	25	100	50	100
NaeI	1	100	75	10	75
NarI	1	100	75	75	75
Nb.BsmI @65°C	3	25	100	100	25
NciI	4	100	25	10	100
NcoI	4	100	100	100	100
NdeI	4	75	100	75	100
NgoMIV	4	100	50	10	100
NheI	2 + BSA	100	100	10	100
NheI-HF™	4 +BSA	100	10	0	100
NlaIII	4 + BSA	25	25	25	100
NlaIV	4 + BSA	10	10	10	100
NotI	3 + BSA	0	50	100	25
NruI	3	0	10	100	10
NsiI	3	10	75	100	25
NspI	2 + BSA	100	100	0	100
PacI	1 + BSA	100	75	10	100
PaeR7I	4	25	100	10	100
PciI	3	50	75	100	50
PflFI	4 + BSA	0	25	25	100
PflMI	3 + BSA	0	100	100	50
PhoI@75°C	3	50	50	100	75
PI-PspI @65°C	U + BSA	0	10	10	10
PI-SceI	U + BSA	0	0	0	0
PleI	4	100	100	75	100
PmeI	4 + BSA	0	50	10	100
PmlI	1 + BSA	100	75	0	75
PpuMI	4	0	25	0	100
PshAI	4 + BSA	50	75	10	100
PsiI	4	10	100	10	100
PspGI @75°C	2	75	100	50	100
PspOMI	4	25	25	10	100
PspXI	2 + BSA	10	100	10	50
PstI	3 + BSA	75	75	100	50
PvuI	3 + BSA	10	75	100	10
PvuII	2	100	100	100	100
PvuII-HF™	4	0	25	0	100
RsaI	1	100	100	50	100
RsrII	4	25	75	10	100
SacI	1 + BSA	100	50	10	100
SacII	4	25	75	10	100
SalI	3 + BSA	0	0	100	0
SalI-HF™	4	10	100	100	100
SapI	4	75	50	0	100
Sau3AI	1 + BSA	100	50	10	100
Sau96I	4	50	100	100	100
SbfI	4	75	50	0	100
ScaI	3	100	100	100^dd	25
ScaI-HF™	4	100	100	10	100
ScrFI	4	100	100	100	100
SexAI	3 + BSA	25	25	100	100
SfaNI	3	0	75	100	25
SfcI	4 + BSA	75	50	10	100
SfiI @50°C	2 + BSA	0	100	10	100
SfoI	2	25	100	50	50
SgrAI	4	100	50	10	100
SmaI @25°C	4	0	0	0	100
SmlI @55°C	4 + BSA	25	75	25	100
SnaBI	4 + BSA	25	50	25	100^dd
SpeI	2 + BSA	75	100	25	75
SphI	2	100	100	50	100
SphI-HF™	4	50	25	10	100
SspI	U	50	100	50	50
StuI	2	100	100	50	100
StyI	3 + BSA	25	75	100	10
StyD4I	2	10	100	100	100
SwaI @25°C	3 + BSA	10	10	100	10
Taq I @65°C	3 + BSA	50	75	100	75
TfiI @65°C	3	100	100	100^dd	100
TliI @75°C	3 + BSA	25	25	100	10
TseI @65°C	3	75	100	100	75
Tsp45I @65°C	1 + BSA	100	25	0	75
Tsp509I @65°C	1	100	100	100	NR
TspMI @75°C	4	50	75	50	100
TspRI @65°C	4 + BSA	25	50	25	100
Tth111I @65°C	1	100	25	25	100
XbaI	2 + BSA	0	100	75	75
XcmI	2	10	100	50	50
XhoI	2 + BSA	75	100	100	100
XmaI	4 + BSA	25	50	0	100
XmnI	2 + BSA	100	100	75	100
ZraI	1	100	25	10	50

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