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Home > Technical Reference > Restriction Endonucleases > Simplified Buffer System

The Switch to NEBuffer 4 and a Simplified Buffer System

On November 1st 2008, New England Biolabs changed the recommended buffer for 54 of our restriction enzymes to NEBuffer 4. With this change, NEB provides 162 restriction enzymes in a single buffer.

Restriction enzymes are generally supplied with a single reaction buffer, although in many instances the enzyme exhibits optimal activity in more than one buffer. A review of our restriction enzymes indicated that NEBuffer 4 was the most commonly recommended buffer and that many of those enzymes supplied in NEBuffers 1,2, and 3 showed similar activity in NEBuffer 4. These enzymes were subsequently re-assayed and thoroughly tested for star activity, stability, nuclease activity and other noted properties. Those that passed testing were switched to NEBuffer 4. This change will improve efficiency and ease of use especially when performing double digests. Please click here for FAQs about this NEBuffer 4 switch.

In addition NEB scientists have been able to engineer enzymes for greatly reduced star activity. These new High Fidelity enzymes, noted by icon

and -HF following the enzyme name all recommend and are supplied with NEBuffer 4.

Please also check these additional information about:
NEBuffer Activity | Double Digestion | Time-Saver Enzymes | High Fidelity (HF) Restriction Enzymes

Enzymes Supplied in NEBuffer 4 (Click for the Chart Legend, Icon Description)

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

Enzyme	Supplied NEBuffer	% Activity in NEBuffers				Heat Inactiva- tion °C	Incuba. Temp. °C	Diluent
Enzyme	Supplied NEBuffer	1	2	3	4	Heat Inactiva- tion °C	Incuba. Temp. °C	Diluent
AatII	NEBuffer 4	0	50	50	100	65	37	A
AccI	NEBuffer 4	50	50	10	100	80	37	A
AclI	NEBuffer 4 + BSA	10	10	0	100	No	37	B
AcuI	NEBuffer 4 + SAM	50	100	50	100	65	37	B
AfeI	NEBuffer 4	25	50	25	100	65	37	B
AflII	NEBuffer 4 + BSA	50	100	25	100	65	37	A
AgeI-HF™	NEBuffer 4 + BSA	100	50	10	100	65	37	A
AhdI	NEBuffer 4 + BSA	25	75	0	100	65	37	A
AleI	NEBuffer 4	10	25	10	100	65	37	A
AluI	NEBuffer 4	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
AscI	NEBuffer 4	0	10	10	100	65	37	A
AsiSI	NEBuffer 4 + BSA	50	100	100	100	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 4	100	100	50	100	80	37	B
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
BaeI	NEBuffer 4 + BSA + SAM	50	100	50	100	65	25	A
BamHI-HF^™	NEBuffer 4	100	50	10	100	No	37	A
BanI	NEBuffer 4 + BSA	50	100	50	100	65	37	A
BanII	NEBuffer 4	100	10	50	100	80	37	A
BbvCI	NEBuffer 4	50	100	10	100	80	37	A
BciVI	NEBuffer 4	100	50	0	100	65	37	C
BfaI	NEBuffer 4	75	50	10	100	80	37	B
BfuCI	NEBuffer 4 + BSA	100	50	25	100	80	37	B
BlpI	NEBuffer 4	50	100	10	100	No	37	A
BpuEI	NEBuffer 4 + SAM	50	100	10	100	65	37	B
BsaAI	NEBuffer 4	100	100	100	100	No	37	C
BsaBI	NEBuffer 4	50	100	75	100	80	60	B
BsaHI	NEBuffer 4 + BSA	50	100	100	100	80	37	A
BsaI	NEBuffer 4 + BSA	75	75	100	100	65	37	B
BsaI-HF™	NEBuffer 4 + BSA	25	25	10	100	65	37	B
BsaJI	NEBuffer 4	100	100	100	100	80	60	A
BsaWI	NEBuffer 4 + BSA	50	100	50	100	80	60	A
BsaXI	NEBuffer 4	75	100	10	100	No	37	B
BseRI	NEBuffer 4	100	100	75	100	65	37	A
BsgI	NEBuffer 4 + SAM	50	75	50	100	65	37	B
BsiEI	NEBuffer 4 + BSA	50	100	10	100	80	60	A
BsiHKAI	NEBuffer 4 + BSA	50	100	100	100	No	65	B
BsmAI	NEBuffer 4	50	100	100	100	80	55	B
BsmFI	NEBuffer 4 + BSA	25	50	50	100	80	65	A
BsmI	NEBuffer 4	75	100	75	100	80	65	A
Bsp1286I	NEBuffer 4 + BSA	25	25	25	100	65	37	A
BspCNI	NEBuffer 4 + BSA + SAM	100	75	10	100	80	25	A
BspDI	NEBuffer 4	25	75	50	100	65	37	A
BspHI	NEBuffer 4	10	100	50	100	65	37	A
BspQI	NEBuffer 4	10	50	100	100	80	50	B
BsrBI	NEBuffer 4	50	100	100	100	80	37	A
BsrFI	NEBuffer 4	10	100	100	100	No	37	C
BstAPI	NEBuffer 4 + BSA	25	100	100	100	80	60	A
BstBI	NEBuffer 4	75	50	25	100	No	65	A
BstUI	NEBuffer 4	100	100	50	100	No	60	A
BstZ17I	NEBuffer 4	NR	NR	100	100	No	37	B
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 4	50	75	100	100	65	37	B
ClaI	NEBuffer 4 + BSA	10	50	50	100	65	37	A
CspCI	NEBuffer 4 + SAM	10	100	10	100	65	37	A
CviAII	NEBuffer 4 + BSA	75	25	10	100	65	25	C
CviKI-1	NEBuffer 4	10	50	25	100	80	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
DpnI	NEBuffer 4	100	100	75	100	80	37	B
DraI	NEBuffer 4	75	75	50	100	65	37	A
DrdI	NEBuffer 4 + BSA	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 4	100	100	50	100	80	37	A
EagI-HF^™	NEBuffer 4	25	100	100	100	65	37	B
EarI	NEBuffer 4	100	100	50	100	65	37	A
Eco53kI	NEBuffer 4	100	50	25	100	65	37	A
EcoNI	NEBuffer 4	100	100	75	100	65	37	A
EcoO109I	NEBuffer 4 + BSA	100	100	75	100	65	37	A
EcoRI-HF^™	NEBuffer 4	10	100	0	100	65	37	C
EcoRV-HF^™	NEBuffer 4	25	100	100	100	65	37	B
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
FauI	NEBuffer 4	100	50	10	100	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	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 4	50	100	25	100	80	37	A
HhaI	NEBuffer 4 + BSA	75	100	100	100	65	37	A
HinfI	NEBuffer 4	75	100	75	100	80	37	A
HinP1I	NEBuffer 4	100	100	100	100	65	37	A
HpaI	NEBuffer 4	25	25	10	100	No	37	A
HphI	NEBuffer 4	NR	75	0	100	65	37	B
Hpy166II	NEBuffer 4	100	100	50	100	65	37	C
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
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
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
KasI	NEBuffer 4 + BSA	25	100	0	100	65	37	B
KpnI-HF™	NEBuffer 4	100	25	0	100	No	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
MboI	NEBuffer 4	75	100	100	100	65	37	A
MboII	NEBuffer 4	100	100	50	100	65	37	C
MfeI	NEBuffer 4	75	50	10	100	65	37	A
MfeI-HF^™	NEBuffer 4	75	50	10	100	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 4 + BSA	75	100	50	100	65	37	B
MscI	NEBuffer 4	75	75	75	100	65	37	B
MseI	NEBuffer 4 + BSA	75	100	75	100	65	37	A
MslI	NEBuffer 4	100	100	25	100	65	37	A
MspA1I	NEBuffer 4 + BSA	50	100	50	100	65	37	B
MspI	NEBuffer 4	75	100	50	100	No	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
NaeI	NEBuffer 4	100	75	10	100	65	37	A
NarI	NEBuffer 4	100	75	75	100	65	37	A
Nb.BtsI	NEBuffer 4 + BSA	75	100	75	100	80	37	A
Nb.BtsI	NEBuffer 4 + BSA	75	100	75	100	80	37	A
NciI	NEBuffer 4	100	25	10	100	No	37	A
NcoI-HF^™	NEBuffer 4	50	100	10	100	80	37	B
NdeI	NEBuffer 4	75	100	75	100	65	37	A
NgoMIV	NEBuffer 4	100	50	10	100	80	37	A
NheI-HF^™	NEBuffer 4 + BSA	100	10	0	100	80	37	C
NlaIII	NEBuffer 4 + BSA	25	25	25	100	65	37	B
NlaIV	NEBuffer 4 + BSA	10	10	10	100	65	37	B
NmeAIII	NEBuffer 4 + SAM	10	10	0	100	80	37	B
NotI-HF^™	NEBuffer 4 + BSA	25	100	25	100	65	37	A
Nt.BbvCI	NEBuffer 4	50	100	10	100	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.BsmAI	NEBuffer 4	100	50	10	100	65	37	A
Nt.CviPII	NEBuffer 4	25	100	50	100	65	37	A
Nt.CviPII	NEBuffer 4	25	100	50	100	65	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
PaeR7I	NEBuffer 4	25	100	10	100	No	37	A
PflFI	NEBuffer 4 + BSA	0	25	25	100	65	37	A
PleI	NEBuffer 4	100	100	75	100	65	37	A
PmeI	NEBuffer 4 + BSA	0	50	10	100	65	37	A
PpuMI	NEBuffer 4	0	25	0	100	No	37	B
PshAI	NEBuffer 4 + BSA	50	75	10	100	65	37	A
PsiI	NEBuffer 4	10	100	10	100	65	37	B
PspGI	NEBuffer 4	75	100	50	100	No	75	A
PspOMI	NEBuffer 4	25	25	10	100	65	37	B
PspXI	NEBuffer 4	0	100	10	100	80	37	B
PstI-HF™	NEBuffer 4	10	75	50	100	80	37	C
PvuII-HF^™	NEBuffer 4	0	25	0	100	80	37	B
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
RsaI	NEBuffer 4	100	100	50	100	65	37	A
RsrII	NEBuffer 4	25	75	10	100	65	37	C
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
SacI-HF^™	NEBuffer 4 + BSA	100	50	10	100	65	37	A
SacII	NEBuffer 4	25	75	10	100	65	37	A
SalI-HF^™	NEBuffer 4	10	100	100	100	65	37	A
SapI	NEBuffer 4	75	50	0	100	65	37	C
Sau96I	NEBuffer 4	50	100	100	100	80	37	A
SbfI	NEBuffer 4	75	50	0	100	65	37	A
SbfI-HF^™	NEBuffer 4	50	25	0	100	65	37	B
ScaI-HF^™	NEBuffer 4	100	100	10	100	65	37	B
ScrFI	NEBuffer 4	100	100	100	100	65	37	C
SexAI	NEBuffer 4 + BSA	100	75	50	100	65	37	A
SfcI	NEBuffer 4 + BSA	75	50	10	100	65	37	B
SfiI	NEBuffer 4 + BSA	0	100	10	100	No	50	C
SfoI	NEBuffer 4	25	100	50	100	No	37	B
SgrAI	NEBuffer 4	100	50	10	100	65	37	A
SmaI	NEBuffer 4	0	0	0	100	65	25	A
SmlI	NEBuffer 4 + BSA	25	75	25	100	No	55	A
SnaBI	NEBuffer 4 + BSA	25	50	25	100 ^dd	80	37	A
SpeI	NEBuffer 4 + BSA	75	100	25	100	80	37	C
SphI-HF^™	NEBuffer 4	50	25	10	100	65	37	B
SspI-HF^™	NEBuffer 4	25	100	0	100	65	37	B
StuI	NEBuffer 4	100	100	50	100	65	37	A
StyI-HF™	NEBuffer 4	25	100	25	100	65	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
Taq^αI	NEBuffer 4 + BSA	50	75	100	100	80	65	B
TfiI	NEBuffer 4	100	100	100	100	No	65	C
TseI	NEBuffer 4	75	100	100	100	No	65	B
TspMI	NEBuffer 4	50	75	50	100	No	75	B
TspRI	NEBuffer 4 + BSA	25	50	25	100	No	65	B
Tth111I	NEBuffer 4	50	25	25	100	No	65	B
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
XbaI	NEBuffer 4 + BSA	0	100	75	100	65	37	A
XhoI	NEBuffer 4 + BSA	75	100	100	100	65	37	A
XmaI	NEBuffer 4 + BSA	25	50	0	100	65	37	A
XmnI	NEBuffer 4 + BSA	100	100	50	100	65	37	A
Enzyme top	Supplied NEBuffer	% in B1	% in B2	% in B3	% in B4	Heat Inactiva.	Incuba. Temp.	Diluent
ZraI	NEBuffer 4	100	25	10	100	No	37	B

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

FAQs about NEBuffer 4 switch
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Q. The NEB catalog has historically stated that activity in NEBuffer4 is less than 100% yet this enzyme is now supplied with NEBuffer4. What have you changed?
A. All our restriction enzymes were re-assayed in both NEBuffer4 vs. the previously supplied NEBuffer. In a few cases minor formulation changes were required to bring the activity to 100% in NEBuffer 4.

Q. Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
A. In general the properties of the restriction enzyme remain the same although for some enzymes, some minor changes in heat inactivation, Time-Saver™ qualification, etc. were observed. All the updated information can be found on the supplied data card as well as at www.neb.com.

Q. If I have an old tube of enzyme, what NEBuffer should I use?
A. Our restriction enzymes are color coded on the label for the appropriate NEBuffer and can either be used with the previously supplied NEBuffer or with NEBuffer 4. To ensure 100% cleavage in NEBuffer 4, additional units of enzyme and/or longer incubation time may be necessary.

Q. Will the new enzyme work in the originally supplied NEBuffer?
A. Yes it will. In all instances the restriction enzyme will have 100% activity in the originally supplied NEBuffer.

Q.Why is NEB switching this restriction enzyme to NEBuffer 4?
A. Our main goal is to simplify the NEBuffer system so that the majority of restriction enzymes are compatible in a single buffer. We now supply 162 restriction enzymes with NEBuffer 4 including all the new High Fidelity (HF) restriction enzymes.