Home Resources 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 >210 restriction enzymes that cut in a single buffer, rCutSmart™ . 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 and star activity. For information on whether or not more than 1-site is required for cleavage (digestion), please refer to the individual product page.

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.

To address the increased need for BSA-free reagents, all reaction NEBuffers are now BSA-free. We are also in the process of transitioning our enzyme formulations to contain rAlbumin.

Legend

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

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

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
r1.1 r2.1 r3.1 rCutSmart
AatII    GACGT/C rCutSmart™ Buffer <10 50* 50 100 80°C 37°C B λ DNA
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-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 λ DNA
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 37°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-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 50 100 50 100 65°C 37°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 37°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-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 1
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 80°C 55°C B ΦX174 DNA
BsiWI-HF®     C/GTACG rCutSmart™ Buffer 50 100 10 100 80°C 37°C B ΦX174 DNA
BslI     CCNNNNN/NNGG rCutSmart™ Buffer 50 75 100 100 No 37°C A λ DNA b
BsmAI    GTCTC(1/5) rCutSmart™ Buffer 50 100 100 100 No 37°C B λ DNA
BsmBI-v2     CGTCTC NEBuffer™ r3.1 <10 50 100 25 80°C 55°C B λ DNA
BsmFI    GGGAC(10/14) rCutSmart™ Buffer 25 50 50 100 80°C 37°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
BspQI-HF® §    GCTCTTC(1/4) rCutSmart™ Buffer 10 50 25 100 80°C 37°C B λ DNA
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 37°C A λ DNA 3, d
BsrFI-v2     R/CCGGY rCutSmart™ Buffer 25 25 0 100 No 37°C C pBR322 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 37°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-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 rCutSmart™ Buffer 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
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
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 rCutSmart™ Buffer 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
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
Hpy99I   CGWCG/ rCutSmart™ Buffer 50 10 <10 100 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
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-HF®    GGTAC/C rCutSmart™ Buffer 100 25 <10 100 No 37°C A pXba DNA
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-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-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 + Activator <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(none/-2) rCutSmart™ Buffer 25 100 100 100 80°C 37°C A supercoiled plasmid DNA e
Nb.BsmI GAATGC(none/-1) NEBuffer™ r3.1 <10 50 100 10 80°C 65°C A supercoiled plasmid pBR322 DNA e
Nb.BsrDI GCAATG(none/0) rCutSmart™ Buffer 25 100 100 100 80°C 37°C A supercoiled pUC19 DNA e
Nb.BssSI CACGAG(none/-1) NEBuffer™ r3.1 10 100 100 25 No 37°C B supercoiled pUC19 DNA e
Nb.BtsI GCAGTG(none/0) 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-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/none) rCutSmart™ Buffer 10 100 100 100 80°C 37°C A pUC101 DNA (dam-/dcm-) e
Nt.BbvCI   CCTCAGC(-5/none) rCutSmart™ Buffer 50 100 10 100 80°C 37°C A supercoiled plasmid DNA e
Nt.BsmAI   GTCTC(1/none) rCutSmart™ Buffer 100 50 10 100 65°C 37°C A supercoiled plasmid DNA e
Nt.BspQI GCTCTTC(1/none) NEBuffer™ r3.1 <10 25 100 10 80°C 50°C B supercoiled pUC19 DNA e
Nt.BstNBI GAGTC(4/none) NEBuffer™ r3.1 0 10 100 10 80°C 55°C A T7 DNA e
Nt.CviPII   CCD(-3/none) 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 + Activator 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 + rAlbumin 10 10 10 10 No 65°C B pAKR7 XmnI-linearized Control Plasmid
PI-SceI ATCTATGTCGGGTGCGGAGAAAGAGGTAAT(-15/-19) NEBuffer™ PI-SceI + rAlbumin 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-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-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-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 37°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-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-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
WarmStart® Nt.BstNBI GAGTC(4/none) NEBuffer™ r3.1 0 10 100 25 80°C 55°C A T7 DNA
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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