Home Tools & Resources Usage Guidelines NEBuffer Activity/Performance Chart with Restriction Enzymes

NEBuffer Activity/Performance Chart with Restriction Enzymes

NEB’s restriction enzyme buffer system makes your restriction digests easy and convenient. We are able to offer >215 restriction enzymes that cut in a single buffer, CutSmart^® . This improves ease-of-use, especially when performing double digests. In addition to indicating the performance of each enzyme in the 4 NEBuffers, the chart also indicates ligation and recutting, star activity, and whether or not more than 1-site is required for cleavage.

Should you require information on performing restriction enzyme digestions, please refer to Optimizing Restriction Endonuclease Reactions. You can also receive additional support by contacting info@neb.com.

We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer™ 1.1, 2.1, 3.1 and CutSmart^® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We anticipate that this switch may take as long as 6 months to complete. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Find more details at www.neb.com/BSA-free.

During this transition period, you may receive product with BSA or rAlbumin-containing buffers. NEB has rigorously tested both and has not seen any difference in enzyme performance when using either buffer. Either buffer can be used with your enzyme. All website content will be switched in April to reflect the changes, although you may not receive the new buffer with your product immediately.

Legend

	Not Sensitive		Impaired
	Blocked		Impaired by Overlapping
	Blocked by Overlapping		Impaired by Some Combinations of Overlapping
	Blocked by Some Combinations of Overlapping

Chart Notes | Icon Descriptions | Single Letter Code

Enzyme	Sequence	Supplied NEBuffer	% Activity in NEBuffer				Heat Inac.	Incu. Temp.	Diluent	Dam	Dcm	CpG	Unit Substrate	Notes
	Sequence	Supplied NEBuffer	r1.1	r2.1	r3.1	rCutSmart	Heat Inac.	Incu. Temp.	Diluent	Dam	Dcm	CpG	Unit Substrate	Notes
AatII	GACGT/C	rCutSmart™ Buffer	<10	50*	50	100	80°C	37°C	B				λ DNA
AbaSI	CNNNNNNNNNNN/NNNNNNNNNG	rCutSmart™ Buffer	25	50	50	100	65°C	25°C	C				T4 wild-type phage DNA (fully ghmC-modified)	e
AccI	GT/MKAC	rCutSmart™ Buffer	50	50	10	100	80°C	37°C	A				λ DNA
Acc65I	G/GTACC	NEBuffer™ r3.1	10	75*	100	25	65°C	37°C	A				pBC4 DNA
AciI	CCGC(-3/-1)	rCutSmart™ Buffer	<10	25	100	100	65°C	37°C	A				λ DNA
AclI	AA/CGTT	rCutSmart™ Buffer	<10	<10	<10	100	No	37°C	B				λ DNA
AcuI	CTGAAG(16/14)	rCutSmart™ Buffer	50	100	50	100	65°C	37°C	B				λ DNA	1, b, d
AfeI	AGC/GCT	rCutSmart™ Buffer	25	100	25	100	65°C	37°C	B				pXba DNA
AflII	C/TTAAG	rCutSmart™ Buffer	50	100	10	100	65°C	37°C	A				ΦX174 RF I DNA
AflIII	A/CRYGT	NEBuffer™ r3.1	10	50	100	50	80°C	37°C	B				λ DNA
AgeI ^§	A/CCGGT	NEBuffer™ r1.1	100	75	25	75	65°C	37°C	C				λ DNA
AgeI-HF®	A/CCGGT	rCutSmart™ Buffer	100	50	10	100	65°C	37°C	A				λ DNA
AhdI	GACNNN/NNGTC	rCutSmart™ Buffer	25	25	10	100	65°C	37°C	A				λ DNA	a
AleI-v2	CACNN/NNGTG	rCutSmart™ Buffer	<10	<10	<10	100	65°C	37°C	B
AluI	AG/CT	rCutSmart™ Buffer	25	100	50	100	80°C	37°C	B				λ DNA	b
AlwI	GGATC(4/5)	rCutSmart™ Buffer	50	50	10	100	No	37°C	A				λ DNA (dam-)	1, b, d
AlwNI	CAGNNN/CTG	rCutSmart™ Buffer	10	100	50	100	80°C	37°C	A				λ DNA
ApaI	GGGCC/C	rCutSmart™ Buffer	25	25	<10	100	65°C	25°C	A				pXba DNA
ApaLI	G/TGCAC	rCutSmart™ Buffer	100	100	10	100	No	37°C	A				λ DNA (HindIII digest)
ApeKI	G/CWGC	NEBuffer™ r3.1	25	50	100	10	No	75°C	B				λ DNA
ApoI ^§	R/AATTY	NEBuffer™ r3.1	10	75	100	75	80°C	50°C	A				λ DNA
ApoI-HF	R/AATTY	rCutSmart™ Buffer	10	100	10	100	80°C	37°C	B				λ DNA
AscI	GG/CGCGCC	rCutSmart™ Buffer	<10	10	10	100	80°C	37°C	A				λ DNA
AseI	AT/TAAT	NEBuffer™ r3.1	<10	50*	100	10	65°C	37°C	B				λ DNA	3
AsiSI	GCGAT/CGC	rCutSmart™ Buffer	100	100	25	100	80°C	37°C	B				XhoI digested pXba	2, b
AvaI	C/YCGRG	rCutSmart™ Buffer	<10	100	25	100	80°C	37°C	A				λ DNA
AvaII	G/GWCC	rCutSmart™ Buffer	50	75	10	100	80°C	37°C	A				λ DNA
AvrII	C/CTAGG	rCutSmart™ Buffer	100	50	50	100	No	37°C	B				λ DNA (HindIII digest)
BaeGI	GKGCM/C	NEBuffer™ r3.1	75	75	100	25	80°C	37°C	A				λ DNA
BaeI	(10/15)ACNNNNGTAYC(12/7)	rCutSmart™ Buffer + SAM	50	100	50	100	65°C	25°C	A				λ DNA	e
BamHI ^§	G/GATCC	NEBuffer™ r3.1	75*	100*	100	100*	No	37°C	A				λ DNA	3
BamHI-HF®	G/GATCC	rCutSmart™ Buffer	100	50	10	100	No	37°C	A				λ DNA
BanI	G/GYRCC	rCutSmart™ Buffer	10	25	<10	100	65°C	37°C	A				λ DNA	1
BanII	GRGCY/C	rCutSmart™ Buffer	100	100	50	100	80°C	37°C	A				λ DNA	2
BbsI ^§	GAAGAC(2/6)	NEBuffer™ r2.1	100	100	25	75	65°C	37°C	B				λ DNA
BbsI-HF^®	GAAGAC(2/6)	rCutSmart™ Buffer	10	10	10	100	65°C	37°C	B				λ DNA
BbvCI	CCTCAGC(-5/-2)	rCutSmart™ Buffer	10	100	50	100	No	37°C	B				λ DNA	1, a
BbvI	GCAGC(8/12)	rCutSmart™ Buffer	100	100	25	100	65°C	37°C	B				pBR322 DNA	3
BccI	CCATC(4/5)	rCutSmart™ Buffer	100	50	10	100	65°C	37°C	A				pXba DNA	3, b
BceAI	ACGGC(12/14)	NEBuffer™ r3.1	100*	100*	100	100*	65°C	37°C	A				pBR322 DNA	1
BcgI	(10/12)CGANNNNNNTGC(12/10)	NEBuffer™ r3.1	10	75*	100	50*	65°C	37°C	A				λ DNA	e
BciVI	GTATCC(6/5)	rCutSmart™ Buffer	100	25	<10	100	80°C	37°C	C				λ DNA	b
BclI ^§	T/GATCA	NEBuffer™ r3.1	50	100	100	75	No	50°C	A				λ DNA (dam-)
BclI-HF	T/GATCA	rCutSmart™ Buffer	100	100	10	100	65°C	37°C	B				λ DNA (dam-)
BcoDI	GTCTC(1/5)	rCutSmart™ Buffer	50	75	75	100	No	37°C	B				λ DNA
BfaI	C/TAG	rCutSmart™ Buffer	<10	10	<10	100	80°C	37°C	B				λ DNA	2, b
BfuAI	ACCTGC(4/8)	NEBuffer™ r3.1	<10	25	100	10	65°C	50°C	B				λ DNA	3
BglI	GCCNNNN/NGGC	NEBuffer™ r3.1	10	25	100	10	65°C	37°C	B				λ DNA
BglII	A/GATCT	NEBuffer™ r3.1	10	10	100	<10	No	37°C	A				λ DNA
BlpI	GC/TNAGC	rCutSmart™ Buffer	50	100	10	100	No	37°C	A				λ DNA	d
BmgBI	CACGTC(-3/-3)	NEBuffer™ r3.1	<10	10	100	10	65°C	37°C	B				λ DNA	3, b, d
BmrI	ACTGGG(5/4)	NEBuffer™ r2.1	75	100	75	100*	65°C	37°C	B				λ DNA (HindIII digest)	b
BmtI ^§	GCTAG/C	NEBuffer™ r3.1	100	100	100	100⁺	65°C	37°C	B				pXba DNA	2
BmtI-HF®	GCTAG/C	rCutSmart™ Buffer	50	100	10	100	65°C	37°C	B				pXba DNA
BpmI	CTGGAG(16/14)	NEBuffer™ r3.1	75	100	100	100*	65°C	37°C	B				λ DNA	2
BpuEI	CTTGAG(16/14)	rCutSmart™ Buffer	50*	100	50*	100	65°C	37°C	B				λ DNA	d
Bpu10I	CCTNAGC(-5/-2)	NEBuffer™ r3.1	10	25	100	25	80°C	37°C	B				λ DNA	3, b, d
BsaAI	YAC/GTR	rCutSmart™ Buffer	100	100	100	100	No	37°C	C				λ DNA
BsaBI	GATNN/NNATC	rCutSmart™ Buffer	50	100	75	100	80°C	60°C	B				λ DNA (dam-)	2
BsaHI	GR/CGYC	rCutSmart™ Buffer	50	100	100	100	80°C	37°C	C				λ DNA
BsaI-HF®v2	GGTCTC(1/5)	rCutSmart™ Buffer	100	100	100	100	80°C	37°C	B				pXba DNA
BsaJI	C/CNNGG	rCutSmart™ Buffer	50	100	100	100	80°C	60°C	A				λ DNA
BsaWI	W/CCGGW	rCutSmart™ Buffer	10	100	50	100	80°C	60°C	A				λ DNA
BsaXI	(9/12)ACNNNNNCTCC(10/7)	rCutSmart™ Buffer	50*	100*	10	100	No	37°C	C				λ DNA	e
BseRI	GAGGAG(10/8)	rCutSmart™ Buffer	100	100	75	100	80°C	37°C	A				λ DNA	d
BseYI	CCCAGC(-5/-1)	NEBuffer™ r3.1	10	50	100	50	80°C	37°C	B				λ DNA	d
BsgI	GTGCAG(16/14)	rCutSmart™ Buffer	25	50	25	100	65°C	37°C	B				λ DNA	d
BsiEI	CGRY/CG	rCutSmart™ Buffer	25	50	<10	100	No	60°C	A				λ DNA
BsiHKAI	GWGCW/C	rCutSmart™ Buffer	25	100	100	100	No	65°C	A				λ DNA
BsiWI ^§	C/GTACG	NEBuffer™ r3.1	25	50*	100	25	65°C	55°C	B				ΦX174 DNA
BsiWI-HF^®	C/GTACG	rCutSmart™ Buffer	50	100	10	100	No	37°C	B				ΦX174 DNA
BslI	CCNNNNN/NNGG	rCutSmart™ Buffer	50	75	100	100	No	55°C	A				λ DNA	b
BsmAI	GTCTC(1/5)	rCutSmart™ Buffer	50	100	100	100	No	55°C	B				λ DNA
BsmBI-v2	CGTCTC	NEBuffer™ r3.1	<10	50	100	25	80°C	55°C	B
BsmFI	GGGAC(10/14)	rCutSmart™ Buffer	25	50	50	100	80°C	65°C	A				pBR322 DNA	1
BsmI	GAATGC(1/-1)	rCutSmart™ Buffer	25	100	<10	100	80°C	65°C	A				λ DNA
BsoBI	C/YCGRG	rCutSmart™ Buffer	25	100	100	100	80°C	37°C	A				λ DNA
BspCNI	CTCAG(9/7)	rCutSmart™ Buffer	100	75	10	100	80°C	37°C	A				λ DNA	b
BspDI	AT/CGAT	rCutSmart™ Buffer	25	75	50	100	80°C	37°C	A				λ DNA
BspEI	T/CCGGA	NEBuffer™ r3.1	<10	10	100	<10	80°C	37°C	B				λ DNA (dam-)
BspHI	T/CATGA	rCutSmart™ Buffer	10	50	25	100	80°C	37°C	A				λ DNA
Bsp1286I	GDGCH/C	rCutSmart™ Buffer	25	25	25	100	65°C	37°C	A				λ DNA	3
BspMI	ACCTGC(4/8)	NEBuffer™ r3.1	10	50*	100	10	65°C	37°C	B				λ DNA
BspQI	GCTCTTC(1/4)	NEBuffer™ r3.1	100*	100*	100	100*	80°C	50°C	B				λ DNA	3
BsrBI	CCGCTC(-3/-3)	rCutSmart™ Buffer	50	100	100	100	80°C	37°C	A				λ DNA	d
BsrDI	GCAATG(2/0)	NEBuffer™ r2.1	10	100	75	25	80°C	65°C	A				λ DNA	3, d
BsrFI-v2	R/CCGGY	rCutSmart™ Buffer	25	25	0	100	No	37°C	C				pBR322 DNA
BsrGI ^§	T/GTACA	NEBuffer™ r2.1	25	100	100	25	80°C	37°C	A				λ DNA
BsrGI-HF®	T/GTACA	rCutSmart™ Buffer	10	100	100	100	80°C	37°C	A				λ DNA
BsrI	ACTGG(1/-1)	NEBuffer™ r3.1	<10	50	100	10	80°C	65°C	B				ΦX174 DNA	b
BssHII	G/CGCGC	rCutSmart™ Buffer	100	100	100	100	65°C	50°C	B				λ DNA
BssSI-v2	CACGAG(-5/-1)	rCutSmart™ Buffer	10	25	<10	100	No	37°C	B				λ DNA
BstAPI	GCANNNN/NTGC	rCutSmart™ Buffer	50	100	25	100	80°C	60°C	A				λ DNA	b
BstBI	TT/CGAA	rCutSmart™ Buffer	75	100	10	100	No	65°C	A				λ DNA
BstEII ^§	G/GTNACC	NEBuffer™ r3.1	10	75*	100	75*	No	60°C	A				λ DNA	3
BstEII-HF®	G/GTNACC	rCutSmart™ Buffer	<10	10	<10	100	No	37°C	A				λ DNA
BstNI	CC/WGG	NEBuffer™ r3.1	10	100	100	75	No	60°C	A				λ DNA	a
BstUI	CG/CG	rCutSmart™ Buffer	50	100	25	100	No	60°C	A				λ DNA	b
BstXI	CCANNNNN/NTGG	NEBuffer™ r3.1	<10	50	100	25	80°C	37°C	B				λ DNA	3
BstYI	R/GATCY	NEBuffer™ r2.1	25	100	75	100	No	60°C	A				λ DNA
BstZ17I-HF^®	GTATAC	rCutSmart™ Buffer	100	100	10	100	No	37°C	A				λ DNA
Bsu36I	CC/TNAGG	rCutSmart™ Buffer	25	100	100	100	80°C	37°C	C				λ DNA (HindIII digest)	b
BtgI	C/CRYGG	rCutSmart™ Buffer	50	100	100	100	80°C	37°C	B				pBR322 DNA
BtgZI	GCGATG(10/14)	rCutSmart™ Buffer	10	25	<10	100	80°C	60°C	A				λ DNA	3, b, d
BtsCI	GGATG(2/0)	rCutSmart™ Buffer	10	100	25	100	80°C	50°C	B				λ DNA
BtsIMutI	CAGTG(2/0)	rCutSmart™ Buffer	100	50	10	100	80°C	55°C	A				pUC19 DNA	b
BtsI-v2	GCAGTG(2/0)	rCutSmart™ Buffer	100	100	25	100	No	37°C	A				λ DNA	1
Cac8I	GCN/NGC	rCutSmart™ Buffer	50	75	100	100	65°C	37°C	B				λ DNA	b
ClaI	AT/CGAT	rCutSmart™ Buffer	10	50	50	100	65°C	37°C	A				λ DNA (dam-)
CspCI	(11/13)CAANNNNNGTGG(12/10)	rCutSmart™ Buffer	10	100	10	100	65°C	37°C	A				λ DNA	e
CviAII	C/ATG	rCutSmart™ Buffer	50	50	10	100	65°C	25°C	C				λ DNA
CviKI-1	RG/CY	rCutSmart™ Buffer	25	100	100	100	No	37°C	A				pBR322 DNA	1, b
CviQI	G/TAC	NEBuffer™ r3.1	75	100*	100	75*	No	25°C	C				λ DNA	b
DdeI	C/TNAG	rCutSmart™ Buffer	75	100	100	100	65°C	37°C	B				λ DNA
DpnI	GA/TC	rCutSmart™ Buffer	100	100	75	100	80°C	37°C	B				pBR322 DNA (dam methylated)	b
DpnII	/GATC	NEBuffer™ DpnII	25	25	100*	25	65°C	37°C	B				λ DNA (dam-)
DraI	TTT/AAA	rCutSmart™ Buffer	75	75	50	100	65°C	37°C	A				λ DNA
DraIII-HF®	CACNNN/GTG	rCutSmart™ Buffer	<10	50	10	100	No	37°C	B				λ DNA	b
DrdI	GACNNNN/NNGTC	rCutSmart™ Buffer	25	50	10	100	65°C	37°C	A				pUC19 DNA	3
EaeI	Y/GGCCR	rCutSmart™ Buffer	10	50	<10	100	65°C	37°C	A				λ DNA	b
EagI-HF®	C/GGCCG	rCutSmart™ Buffer	25	100	100	100	65°C	37°C	B				pXba DNA
EarI	CTCTTC(1/4)	rCutSmart™ Buffer	50	10	<10	100	65°C	37°C	B				λ DNA	b, d
EciI	GGCGGA(11/9)	rCutSmart™ Buffer	100	50	50	100	65°C	37°C	A				λ DNA	2
Eco53kI	GAG/CTC	rCutSmart™ Buffer	100	100	<10	100	65°C	37°C	A				pXba DNA	3, b
EcoNI	CCTNN/NNNAGG	rCutSmart™ Buffer	50	100	75	100	65°C	37°C	A				λ DNA	b
EcoO109I	RG/GNCCY	rCutSmart™ Buffer	50	100	50	100	65°C	37°C	A				λ DNA (HindIII digest)	3
EcoP15I	CAGCAG(25/27)	NEBuffer™ r3.1 + ATP	75	100	100	100	65°C	37°C	A				pUC19 DNA	e
EcoRI ^§	G/AATTC	NEBuffer™ EcoRI/SspI	25	100*	50	50*	65°C	37°C	C				λ DNA
EcoRI-HF®	G/AATTC	rCutSmart™ Buffer	10	100	<10	100	65°C	37°C	C				λ DNA
EcoRV ^§	GAT/ATC	NEBuffer™ r3.1	10	50	100	10	80°C	37°C	A				λ DNA
EcoRV-HF®	GAT/ATC	rCutSmart™ Buffer	25	100	100	100	65°C	37°C	B				λ DNA
Esp3I	CGTCTC(1/5)	rCutSmart™ Buffer	100	100	<10	100	65°C	37°C	B				λ DNA
FatI	/CATG	NEBuffer™ r2.1	10	100	50	50	80°C	55°C	A				pUC19 DNA
FauI	CCCGC(4/6)	rCutSmart™ Buffer	100	50	10	100	65°C	55°C	A				λ DNA	3, b, d
Fnu4HI	GC/NGC	rCutSmart™ Buffer	<10	<10	<10	100	No	37°C	A				λ DNA	a
FokI	GGATG(9/13)	rCutSmart™ Buffer	100	100	75	100	65°C	37°C	A				λ DNA	3, b, d
FseI	GGCCGG/CC	rCutSmart™ Buffer	100	75	<10	100	65°C	37°C	B				pBC4 DNA
FspEI	CC(12/16)	rCutSmart™ Buffer	<10	<10	<10	100	80°C	37°C	B				pBR322 (dcm+) DNA	1, e
FspI	TGC/GCA	rCutSmart™ Buffer	10	100	10	100	No	37°C	C				λ DNA	b
HaeII	RGCGC/Y	rCutSmart™ Buffer	25	100	10	100	80°C	37°C	A				λ DNA
HaeIII	GG/CC	rCutSmart™ Buffer	50	100	25	100	80°C	37°C	A				λ DNA
HgaI	GACGC(5/10)	NEBuffer™ r1.1	100	100	25	100*	65°C	37°C	A				ΦX174 DNA	1
HhaI	GCG/C	rCutSmart™ Buffer	25	100	100	100	65°C	37°C	A				λ DNA
HincII	GTY/RAC	NEBuffer™ r3.1	25	100	100	100	65°C	37°C	B				λ DNA
HindIII ^§	A/AGCTT	NEBuffer™ r2.1	25	100	50	50	80°C	37°C	B				λ DNA	2
HindIII-HF®	A/AGCTT	rCutSmart™ Buffer	10	100	10	100	80°C	37°C	B				λ DNA
HinfI	G/ANTC	rCutSmart™ Buffer	50	100	100	100	80°C	37°C	A				λ DNA
HinP1I	G/CGC	rCutSmart™ Buffer	100	100	100	100	65°C	37°C	A				λ DNA
HpaI	GTT/AAC	rCutSmart™ Buffer	<10	75*	25	100	No	37°C	A				λ DNA	1
HpaII	C/CGG	rCutSmart™ Buffer	100	50	<10	100	80°C	37°C	A				λ DNA
HphI	GGTGA(8/7)	rCutSmart™ Buffer	50	50	<10	100	65°C	37°C	B				λ DNA	1, b, d
HpyAV	CCTTC(6/5)	rCutSmart™ Buffer	100	100	25	100	65°C	37°C					λ DNA	3, b, d
HpyCH4III	ACN/GT	rCutSmart™ Buffer	100	25	<10	100	65°C	37°C	A				λ DNA	b
HpyCH4IV	A/CGT	rCutSmart™ Buffer	100	50	25	100	65°C	37°C	A				pUC19 DNA
HpyCH4V	TG/CA	rCutSmart™ Buffer	50	50	25	100	65°C	37°C	A				λ DNA
Hpy188I	TCN/GA	rCutSmart™ Buffer	25	100	50	100	65°C	37°C	A				pBR322 DNA	1, b
Hpy99I	CGWCG/	rCutSmart™ Buffer	50	10	<10	100	65°C	37°C	A				λ DNA
Hpy166II	GTN/NAC	rCutSmart™ Buffer	100	100	50	100	65°C	37°C	C				pBR322 DNA
Hpy188III	TC/NNGA	rCutSmart™ Buffer	100	100	10	100	65°C	37°C	B				pUC19 DNA	3, b
I-CeuI	TAACTATAACGGTCCTAAGGTAGCGAA(-9/-13)	rCutSmart™ Buffer	10	10	10	100	65°C	37°C	B				pBHS ScaI-linearized Control Plasmid
I-SceI	TAGGGATAACAGGGTAAT(-9/-13)	rCutSmart™ Buffer	10	50	25	100	65°C	37°C	B				pGPS2 NotI-linearized Control Plasmid
KasI	G/GCGCC	rCutSmart™ Buffer	50	100	50	100	65°C	37°C	B				pBR322 DNA	3
KpnI ^§	GGTAC/C	NEBuffer™ r1.1	100	75	<10	50*	No	37°C	A				pXba DNA	1
KpnI-HF®	GGTAC/C	rCutSmart™ Buffer	100	25	<10	100	No	37°C	A				pXba DNA
LpnPI	CCDG(10/14)	rCutSmart™ Buffer	<10	<10	<10	100	65°C	37°C	B				pBR322 (dcm+) DNA	1, e
MboI	/GATC	rCutSmart™ Buffer	75	100	100	100	65°C	37°C	A				λ DNA (dam-)
MboII	GAAGA(8/7)	rCutSmart™ Buffer	100*	100	50	100	65°C	37°C	C				λ DNA (dam-)	b
MfeI ^§	C/AATTG	rCutSmart™ Buffer	75	50	10	100	No	37°C	A				λ DNA	2
MfeI-HF®	C/AATTG	rCutSmart™ Buffer	75	25	<10	100	No	37°C	A				λ DNA
MluCI	/AATT	rCutSmart™ Buffer	100	10	10	100	No	37°C	A				λ DNA
MluI ^§	A/CGCGT	NEBuffer™ r3.1	10	50	100	25	80°C	37°C	A				λ DNA
MluI-HF®	A/CGCGT	rCutSmart™ Buffer	25	100	100	100	No	37°C	A				λ DNA
MlyI	GAGTC(5/5)	rCutSmart™ Buffer	50	50	10	100	65°C	37°C	A				λ DNA	b, d
MmeI	TCCRAC(20/18)	rCutSmart™ Buffer	50	100	50	100	65°C	37°C	B				ΦX174 RF I DNA	b, c
MnlI	CCTC(7/6)	rCutSmart™ Buffer	75	100	50	100	65°C	37°C	B				λ DNA	b
MscI	TGG/CCA	rCutSmart™ Buffer	25	100	100	100	80°C	37°C	C				λ DNA
MseI	T/TAA	rCutSmart™ Buffer	75	100	75	100	65°C	37°C	A				λ DNA
MslI	CAYNN/NNRTG	rCutSmart™ Buffer	50	50	<10	100	80°C	37°C	A				λ DNA
MspA1I	CMG/CKG	rCutSmart™ Buffer	10	50	10	100	65°C	37°C	B				λ DNA
MspI	C/CGG	rCutSmart™ Buffer	75	100	50	100	No	37°C	A				λ DNA
MspJI	CNNR(9/13)	rCutSmart™ Buffer	<10	<10	<10	100	65°C	37°C	B				pBR322 (dcm+) DNA	1, e
MwoI	GCNNNNN/NNGC	rCutSmart™ Buffer	<10	100	100	100	No	60°C	B				λ DNA
NaeI	GCC/GGC	rCutSmart™ Buffer	25	25	<10	100	No	37°C	A				pXba DNA	b
NarI	GG/CGCC	rCutSmart™ Buffer	100	100	10	100	65°C	37°C	A				pXba DNA
Nb.BbvCI	CCTCAGC	rCutSmart™ Buffer	25	100	100	100	80°C	37°C	A				supercoiled plasmid DNA	e
Nb.BsmI	GAATGC	NEBuffer™ r3.1	<10	50	100	10	80°C	65°C	A				supercoiled plasmid pBR322 DNA	e
Nb.BsrDI	GCAATG	rCutSmart™ Buffer	25	100	100	100	80°C	65°C	A				supercoiled pUC19 DNA	e
Nb.BssSI	CACGAG	NEBuffer™ r3.1	10	100	100	25	No	37°C	B				supercoiled pUC19 DNA	e
Nb.BtsI	GCAGTG	rCutSmart™ Buffer	75	100	75	100	80°C	37°C	A				supercoiled pUC101 DNA (dam-/dcm-)	e
NciI	CC/SGG	rCutSmart™ Buffer	100	25	10	100	No	37°C	A				λ DNA	b
NcoI ^§	C/CATGG	NEBuffer™ r3.1	100	100	100	100⁺	80°C	37°C	A				λ DNA
NcoI-HF®	C/CATGG	rCutSmart™ Buffer	50	100	10	100	80°C	37°C	B				λ DNA
NdeI	CA/TATG	rCutSmart™ Buffer	75	100	100	100	65°C	37°C	A				λ DNA
NgoMIV	G/CCGGC	rCutSmart™ Buffer	100	50	10	100	No	37°C	A				pXba DNA	1
NheI-HF®	G/CTAGC	rCutSmart™ Buffer	100	25	10	100	80°C	37°C	C				λ DNA (HindIII digest)
NlaIII	CATG/	rCutSmart™ Buffer	<10	<10	<10	100	65°C	37°C	B				ΦX174 RF I DNA
NlaIV	GGN/NCC	rCutSmart™ Buffer	10	10	10	100	65°C	37°C	B				pBR322 DNA
NmeAIII	GCCGAG(21/19)	rCutSmart™ Buffer	10	10	<10	100	65°C	37°C	B				ΦX174 RF I DNA	c
NotI ^§	GC/GGCCGC	NEBuffer™ r3.1	<10	50	100	25	65°C	37°C	C				pBC4 DNA
NotI-HF®	GC/GGCCGC	rCutSmart™ Buffer	25	100	25	100	65°C	37°C	A				pBC4 DNA
NruI ^§	TCG/CGA	NEBuffer™ r3.1	<10	10	100	10	No	37°C	A				λ DNA	b
NruI-HF®	TCG/CGA	rCutSmart™ Buffer	0	25	50	100	No	37°C	A				λ DNA
NsiI ^§	ATGCA/T	NEBuffer™ r3.1	10	75	100	25	65°C	37°C	B				λ DNA
NsiI-HF®	ATGCA/T	rCutSmart™ Buffer	<10	20	<10	100	80°C	37°C	B				λ DNA
NspI	RCATG/Y	rCutSmart™ Buffer	100	100	<10	100	65°C	37°C	A				λ DNA
Nt.AlwI	GGATC(4/-5)	rCutSmart™ Buffer	10	100	100	100	80°C	37°C	A				pUC101 DNA (dam-/dcm-)	e
Nt.BbvCI	CCTCAGC(-5/-7)	rCutSmart™ Buffer	50	100	10	100	80°C	37°C	A				supercoiled plasmid DNA	e
Nt.BsmAI	GTCTC(1/-5)	rCutSmart™ Buffer	100	50	10	100	65°C	37°C	A				supercoiled plasmid DNA	e
Nt.BspQI	GCTCTTC(1/-7)	NEBuffer™ r3.1	<10	25	100	10	80°C	50°C	B				supercoiled pUC19 DNA	e
Nt.BstNBI	GAGTC(4/-5)	NEBuffer™ r3.1	0	10	100	10	80°C	55°C	A				T7 DNA	e
Nt.CviPII	(0/-1)CCD	rCutSmart™ Buffer	10	100	25	100	65°C	37°C	A				pUC19 DNA	e
PacI	TTAAT/TAA	rCutSmart™ Buffer	100	75	10	100	65°C	37°C	A				pNEB193 DNA
PaeR7I	C/TCGAG	rCutSmart™ Buffer	25	100	10	100	No	37°C	A				λ DNA (HindIII digest)
PaqCI	CACCTGC(4/8)	rCutSmart™ Buffer	10	100	10	100	65°C	37°C	B				λ DNA	1
PciI	A/CATGT	NEBuffer™ r3.1	50	75	100	50*	80°C	37°C	B				pXba DNA
PflFI	GACN/NNGTC	rCutSmart™ Buffer	25	100	25	100	65°C	37°C	A				pBC4 DNA	b
PflMI	CCANNNN/NTGG	NEBuffer™ r3.1	0	100	100	50	65°C	37°C	A				λ DNA	3, b, d
PI-PspI	TGGCAAACAGCTATTATGGGTATTATGGGT(-13/-17)	NEBuffer™ PI-PspI + BSA	10	10	10	10	No	65°C	B				pAKR7 XmnI-linearized Control Plasmid
PI-SceI	ATCTATGTCGGGTGCGGAGAAAGAGGTAAT(-15/-19)	NEBuffer™ PI-SceI + BSA	10	10	10	10	65°C	37°C	B				pBSvdeX XmnI-linearized Control Plasmid
PleI	GAGTC(4/5)	rCutSmart™ Buffer	25	50	25	100	65°C	37°C	A				λ DNA	b, d
PluTI	GGCGC/C	rCutSmart™ Buffer	100	25	<10	100	65°C	37°C	A				pXba DNA	b
PmeI	GTTT/AAAC	rCutSmart™ Buffer	<10	50	10	100	65°C	37°C	A				λ DNA
PmlI	CAC/GTG	rCutSmart™ Buffer	100	50	<10	100	65°C	37°C	A				λ DNA (HindIII digest) DNA
PpuMI	RG/GWCCY	rCutSmart™ Buffer	<10	<10	<10	100	No	37°C	B				λ DNA (HindIII digest)
PshAI	GACNN/NNGTC	rCutSmart™ Buffer	25	50	10	100	65°C	37°C	A				λ DNA
PsiI-v2	TTA/TAA	rCutSmart™ Buffer	25	50	10	100	65°C	37°C	B				λ DNA	3
PspGI	/CCWGG	rCutSmart™ Buffer	25	100	50	100	No	75°C	A				T7 DNA	3
PspOMI	G/GGCCC	rCutSmart™ Buffer	10	10	<10	100	65°C	37°C	B				pXba DNA
PspXI	VC/TCGAGB	rCutSmart™ Buffer	<10	100	25	100	No	37°C	B				λ DNA (HindIII digest)
PstI ^§	CTGCA/G	NEBuffer™ r3.1	75	75	100	50*	80°C	37°C	C				λ DNA
PstI-HF®	CTGCA/G	rCutSmart™ Buffer	10	75	50	100	No	37°C	C				λ DNA
PvuI ^§	CGAT/CG	NEBuffer™ r3.1	<10	25	100	<10	No	37°C	B				pXba DNA
PvuI-HF®	CGAT/CG	rCutSmart™ Buffer	25	100	100	100	No	37°C	B				pXba DNA
PvuII ^§	CAG/CTG	NEBuffer™ r3.1	50	100	100	100*	No	37°C	B				λ DNA
PvuII-HF®	CAG/CTG	rCutSmart™ Buffer	<10	<10	<10	100	No	37°C	B				λ DNA
RsaI	GT/AC	rCutSmart™ Buffer	25	50	<10	100	No	37°C	A				λ DNA
RsrII	CG/GWCCG	rCutSmart™ Buffer	25	75	10	100	65°C	37°C	C				λ DNA
SacI ^§	GAGCT/C	NEBuffer™ r1.1	100	50	10	100⁺	65°C	37°C	A				λ DNA (HindIII digest)
SacI-HF®	GAGCT/C	rCutSmart™ Buffer	10	50	<10	100	65°C	37°C	A				λ DNA (HindIII digest)
SacII	CCGC/GG	rCutSmart™ Buffer	10	100	10	100	65°C	37°C	A				pXba DNA
SalI ^§	G/TCGAC	NEBuffer™ r3.1	<10	<10	100	<10	65°C	37°C	A				λ DNA (HindIII digest)
SalI-HF®	G/TCGAC	rCutSmart™ Buffer	10	100	100	100	65°C	37°C	A				λ DNA (HindIII digest)
SapI	GCTCTTC(1/4)	rCutSmart™ Buffer	75	50	<10	100	65°C	37°C	B				λ DNA
Sau3AI	/GATC	NEBuffer™ r1.1	100	50	10	100⁺	65°C	37°C	A				λ DNA	b
Sau96I	G/GNCC	rCutSmart™ Buffer	50	100	100	100	65°C	37°C	A				λ DNA
SbfI ^§	CCTGCA/GG	rCutSmart™ Buffer	50	25	<10	100	80°C	37°C	A				λ DNA	3
SbfI-HF®	CCTGCA/GG	rCutSmart™ Buffer	50	25	<10	100	80°C	37°C	B				λ DNA
ScaI-HF®	AGT/ACT	rCutSmart™ Buffer	100	100	10	100	80°C	37°C	B				λ DNA
ScrFI	CC/NGG	rCutSmart™ Buffer	100	100	100	100	65°C	37°C	C				λ DNA	2, a
SexAI	A/CCWGGT	rCutSmart™ Buffer	100	75	50	100	65°C	37°C	A				pBC4 DNA (dcm-)	3, b, d
SfaNI	GCATC(5/9)	NEBuffer™ r3.1	<10	75	100	25	65°C	37°C	B				ΦX174 RF I DNA	3, b
SfcI	C/TRYAG	rCutSmart™ Buffer	75	50	25	100	65°C	37°C	B				λ DNA	3
SfiI	GGCCNNNN/NGGCC	rCutSmart™ Buffer	25	100	50	100	No	50°C	C				pXba DNA
SfoI	GGC/GCC	rCutSmart™ Buffer	50	100	100	100	No	37°C	B				λ DNA (HindIII digest)
SgrAI	CR/CCGGYG	rCutSmart™ Buffer	100	100	10	100	65°C	37°C	A				λ DNA	1
SmaI	CCC/GGG	rCutSmart™ Buffer	<10	<10	<10	100	65°C	25°C	B				λ DNA (HindIII digest)	b
SmlI	C/TYRAG	rCutSmart™ Buffer	25	75	25	100	No	55°C	A				λ DNA	b
SnaBI	TAC/GTA	rCutSmart™ Buffer	50*	50	10	100	80°C	37°C	A				T7 DNA	1
SpeI ^§	A/CTAGT	rCutSmart™ Buffer	75	100	25	100	80°C	37°C	C				pXba-XbaI DNA
SpeI-HF®	A/CTAGT	rCutSmart™ Buffer	25	50	10	100	80°C	37°C	C				pXba-XbaI DNA
SphI ^§	GCATG/C	NEBuffer™ r2.1	100	100	50	100⁺	65°C	37°C	B				λ DNA	2
SphI-HF®	GCATG/C	rCutSmart™ Buffer	50	25	10	100	65°C	37°C	B				λ DNA
SrfI	GCCC/GGGC	rCutSmart™ Buffer	10	50	0	100	65°C	37°C	B				pNEB193-SrfI DNA
SspI ^§	AAT/ATT	NEBuffer™ EcoRI/SspI	50	100	50	50	65°C	37°C	C				λ DNA
SspI-HF®	AAT/ATT	rCutSmart™ Buffer	25	100	<10	100	65°C	37°C	B				λ DNA
StuI	AGG/CCT	rCutSmart™ Buffer	50	100	50	100	No	37°C	A				λ DNA
StyD4I	/CCNGG	rCutSmart™ Buffer	10	100	100	100	65°C	37°C	B				λ DNA
StyI-HF®	C/CWWGG	rCutSmart™ Buffer	25	100	25	100	65°C	37°C	A				λ DNA
SwaI	ATTT/AAAT	NEBuffer™ r3.1	10	10	100	10	65°C	25°C	B				pXba DNA	b, d
TaqI-v2	T/CGA	rCutSmart™ Buffer	50	100	50	100	No	65°C	B				λ DNA
TfiI	G/AWTC	rCutSmart™ Buffer	50	100	100	100	No	65°C	C				λ DNA
TseI	G/CWGC	rCutSmart™ Buffer	75	100	100	100	No	65°C	B				λ DNA	3
Tsp45I	/GTSAC	rCutSmart™ Buffer	100	50	<10	100	No	65°C	A				λ DNA
TspMI	C/CCGGG	rCutSmart™ Buffer	50*	75*	50*	100	No	75°C	B				pBC4 DNA	d
TspRI	NNCASTGNN/	rCutSmart™ Buffer	25	50	25	100	No	65°C	B				λ DNA
Tth111I	GACN/NNGTC	rCutSmart™ Buffer	25	100	25	100	No	65°C	B				pBC4 DNA	b
XbaI	T/CTAGA	rCutSmart™ Buffer	<10	100	75	100	65°C	37°C	A				λ DNA (dam-/Hind III digest)
XcmI	CCANNNNN/NNNNTGG	NEBuffer™ r2.1	10	100	25	100*	65°C	37°C	C				λ DNA	2
XhoI	C/TCGAG	rCutSmart™ Buffer	75	100	100	100	65°C	37°C	A				λ DNA (HindIII digest)	b
XmaI	C/CCGGG	rCutSmart™ Buffer	25	50	<10	100	65°C	37°C	A				pXba DNA	3
XmnI	GAANN/NNTTC	rCutSmart™ Buffer	50	75	<10	100	65°C	37°C	A				λ DNA	b
ZraI	GAC/GTC	rCutSmart™ Buffer	100	25	10	100	80°C	37°C	B				λ DNA

§ An HF version of this enzyme is available.

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

Ligation and Recutting Notes

The following notes appear with any enzymes having ligation efficiencies lower than 100% as assessed by ligation and recutting.

a. Ligation is less than 10%.
b. Ligation is 25% -75%.
c. Recutting after ligation is less than 5%.
d. Recutting after ligation is 50% -75%.
e. Ligation and recutting after ligation is not applicable since the enzyme is either a nicking enzyme, is affected by methylation, or if the enzyme cleaves outside its recognition sequence.

Star Activity Notes

The following notes appear with any enzymes when star activity is a concern.

1. Star Activity may result from extended digestion, high enzyme concentration or a glycerol concentration of > 5%.
2. Star Activity may result from extended digestion.
3. Star Activity may result from a glycerol concentration of > 5%.
*. May exhibit star activity in this buffer.
+. For added flexibility, NEB offers an isoschizomer or HF enzyme, supplied with CutSmart Buffer.

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