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Performance Chart for NEB® Restriction Enzymes
r t AatII GACGT/C CutSmart < 10 50* 50 100 37° 80° B Lambda u
r AbaSI mC(11/9) CutSmart 25 50 50 100 25° 65° C T4 wt Phage e
r t AccI GT/MKAC CutSmart 50 50 10 100 37° 80° A Lambda u
r t Acc65I G/GTACC 3.1 10 75* 100 25 37° 65° A pBC4 I u
r t AciI CCGC(-3/-1) CutSmart < 10 25 100 100 37° 65° A Lambda u d
r t AclI AA/CGTT CutSmart < 10 < 10 < 10 100 37° No B Lambda u
r t AcuI CTGAAG(16/14) CutSmart + SAM 50 100 50 100 37° 65° B Lambda 1, b, d
r AfeI AGC/GCT CutSmart 25 100 25 100 37° 65° B pXba u
r t AflII C/TTAAG CutSmart 50 100 10 100 37° 65° A phiX174
r AflIII A/CRYGT 3.1 10 50 100 50 37° 80° B Lambda
r AgeI A/CCGGT 1.1 100 75 25 75 37° 65° C Lambda u
r t e AgeI-HF A/CCGGT CutSmart 100 50 10 100 37° 65° A Lambda u
r t AhdI GACNNN/NNGTC CutSmart 25 25 10 100 37° 65° A Lambda u a
r AleI-v2 CACNN/NNGTG CutSmart < 10 < 10 < 10 100 37° 80° B Lambda u
r t AluI AG/CT CutSmart 25 100 50 100 37° 80° B Lambda b
r AlwI GGATC(4/5) CutSmart 50 50 10 100 37° No A Lambda dam- i 1, b, d
r t AlwNI CAGNNN/CTG CutSmart 10 100 50 100 37° 80° A Lambda I
r t ApaI GGGCC/C CutSmart 25 25 < 10 100 25° 65° A pXba I u
r t ApaLI G/TGCAC CutSmart 100 100 10 100 37° No A Lambda HindIII u
r t ApeKI G/CWGC 3.1 25 50 100 10 75° No B Lambda u
r t ApoI R/AATTY 3.1 10 75 100 75 50° 80° A Lambda
r t e ApoI-HF R/AATTY CutSmart 10 100 10 100 37° 80° B Lambda
r t AscI GG/CGCGCC CutSmart < 10 10 10 100 37° 80° A Lambda u
r t AseI AT/TAAT 3.1 < 10 50* 100 10 37° 65° B Lambda 3
r AsiSI GCGAT/CGC CutSmart 100 100 25 100 37° 80° B pXba (Xho digested) u 2, b
r t AvaI C/YCGRG CutSmart < 10 100 25 100 37° 80° A Lambda u
r t AvaII G/GWCC CutSmart 50 75 10 100 37° 80° A Lambda I u
r t AvrII C/CTAGG CutSmart 100 50 50 100 37° No B Lambda HindIII
r t BaeI (10/15)AC(N)4GTAYC(12/7) CutSmart + SAM 50 100 50 100 25° 65° A Lambda u e
r t BaeGI GKGCM/C 3.1 75 75 100 25 37° 80° A Lambda
r t BamHI G/GATCC 3.1 75* 100* 100 100* 37° No A Lambda 3
r t e BamHI-HF G/GATCC CutSmart 100 50 10 100 37° No A Lambda
r BanI G/GYRCC CutSmart 10 25 < 10 100 37° 65° A Lambda I u 1
r BanII GRGCY/C CutSmart 100 100 50 100 37° 80° A Lambda 2
r t BbsI GAAGAC(2/6) 2.1 100 100 25 75 37° 65° B Lambda
r t e BbsI-HF GAAGAC(2/6) CutSmart 10 10 10 100 37° 65° B Lambda
r t @ BbvI GCAGC(8/12) CutSmart 100 100 25 100 37° 65° B pBR322 3
r BbvCI CCTCAGC(-5/-2) CutSmart 10 100 50 100 37° No B Lambda u 1, a
r BccI CCATC(4/5) CutSmart 100 50 10 100 37° 65° A pXba 3, b
r BceAI ACGGC(12/14) 3.1 100* 100* 100 100* 37° 65° A pBR322 u 1
r @ BcgI (10/12)CGA(N)6TGC(12/10) 3.1 + SAM 10 75* 100 50* 37° 65° A Lambda i u e
r t BciVI GTATCC(6/5) CutSmart 100 25 < 10 100 37° 80° C Lambda b
r t BclI T/GATCA 3.1 50 100 100 75 50° No A Lambda dam- i
r t e BclI-HF T/GATCA CutSmart 100 100 10 100 37° 65° B Lambda dam- i
r t BcoDI GTCTC(1/5) CutSmart 50 75 75 100 37° No B Lambda u
r BfaI C/TAG CutSmart < 10 10 < 10 100 37° 80° B Lambda 2, b
r t @ BfuAI ACCTGC(4/8) 3.1 < 10 25 100 10 50° 65° B Lambda u 3
r t BglI GCCNNNN/NGGC 3.1 10 25 100 10 37° 65° B Lambda u
r t BglII A/GATCT 3.1 10 10 100 < 10 37° No A Lambda
r t BlpI GC/TNAGC CutSmart 50 100 10 100 37° No A Lambda d
r t BmgBI CACGTC(-3/-3) 3.1 < 10 10 100 10 37° 65° B Lambda u 3, b, d
r BmrI ACTGGG(5/4) 2.1 75 100 75 100* 37° 65° B Lambda HindIII b
r BmtI GCTAG/C 3.1 100 100 100 100 37° 65° B pXba 2
r t e BmtI-HF GCTAG/C CutSmart 50 100 10 100 37° 65° B pXba
r @ BpmI CTGGAG(16/14) 3.1 75 100 100 100 37° 65° B Lambda 2
r Bpu10I CCTNAGC(-5/-2) 3.1 10 25 100 25 37° 80° B Lambda 3, b, d
r t BpuEI CTTGAG(16/14) CutSmart + SAM 50* 100 50* 100 37° 65° B Lambda d
r BsaI GGTCTC(1/5) CutSmart 75* 75 100 100 37° 65° B pXba I u 3
r t e BsaI-HFv2 GGTCTC(1/5) CutSmart 100 100 100 100 37° 80° B pXba I u
r t BsaAI YAC/GTR CutSmart 100 100 100 100 37° No C Lambda u
r BsaBI GATNN/NNATC CutSmart 50 100 75 100 60° 80° B Lambda dam- i u 2
r t BsaHI GR/CGYC CutSmart 50 100 100 100 37° 80° C Lambda I u
r BsaJI C/CNNGG CutSmart 50 100 100 100 60° 80° A Lambda
r t BsaWI W/CCGGW CutSmart 10 100 50 100 60° 80° A Lambda
t BsaXI (9/12)AC(N)5CTCC(10/7) CutSmart 50* 100* 10 100 37° No C Lambda e
r t BseRI GAGGAG(10/8) CutSmart 100 100 75 100 37° 80° A Lambda d
r BseYI CCCAGC(-5/-1) 3.1 10 50 100 50 37° 80° B Lambda u d
r t @ BsgI GTGCAG(16/14) CutSmart + SAM 25 50 25 100 37° 65° B Lambda d
r t BsiEI CGRY/CG CutSmart 25 50 < 10 100 60° No A Lambda u
r BsiHKAI GWGCW/C CutSmart 25 100 100 100 65° No A Lambda
r t BsiWI C/GTACG 3.1 25 50* 100 25 55° 65° B phiX174 u
r t e BsiWI-HF C/GTACG CutSmart 50 100 10 100 37° No B phiX174 u
r t BslI CCNNNNN/NNGG CutSmart 50 75 100 100 55° No A Lambda I u b
r t BsmI GAATGC(1/-1) CutSmart 25 100 < 10 100 65° 80° A Lambda
r t BsmAI GTCTC(1/5) CutSmart 50 100 100 100 55° No B Lambda u
r t BsmBI CGTCTC(1/5) 3.1 10 50* 100 25 55° 80° B Lambda u
r BsmFI GGGAC(10/14) CutSmart 25 50 50 100 65° 80° A pBR322 I u 1
r t BsoBI C/YCGRG CutSmart 25 100 100 100 37° 80° A Lambda
r t Bsp1286I GDGCH/C CutSmart 25 25 25 100 37° 65° A Lambda 3
r t BspCNI CTCAG(9/7) CutSmart + SAM 100 75 10 100 25° 80° A Lambda b
r BspDI AT/CGAT CutSmart 25 75 50 100 37° 80° A Lambda i u
r t BspEI T/CCGGA 3.1 < 10 10 100 < 10 37° 80° B Lambda dam- i u
r t BspHI T/CATGA CutSmart < 10 50 25 100 37° 80° A Lambda i
r @ BspMI ACCTGC(4/8) 3.1 10 50* 100 10 37° 65° B Lambda
r t BspQI GCTCTTC(1/4) 3.1 100* 100* 100 100* 50° 80° B Lambda 3
t BsrI ACTGG(1/-1) 3.1 < 10 50 100 10 65° 80° B phiX174 b
r t BsrBI CCGCTC(-3/-3) CutSmart 50 100 100 100 37° 80° A Lambda u d
r t BsrDI GCAATG(2/0) 2.1 10 100 75 25 65° 80° A Lambda 3, d
r t e BsrFI-v2 R/CCGGY CutSmart 25 25 0 100 37° No C pBR322 u
r t BsrGI T/GTACA 2.1 25 100 100 25 37° 80° A Lambda
r t e BsrGI-HF T/GTACA CutSmart 10 100 100 100 37° 80° A Lambda
r t BssHII G/CGCGC CutSmart 100 100 100 100 50° 65° B Lambda u
r t e BssSI-v2 CACGAG(-5/-1) CutSmart 10 25 < 10 100 37° No B Lambda
r BstAPI GCANNNN/NTGC CutSmart 50 100 25 100 60° 80° A Lambda u b
r t BstBI TT/CGAA CutSmart 75 100 10 100 65° No A Lambda u
r t BstEII G/GTNACC 3.1 10 75* 100 75* 60° No A Lambda 3
r t e BstEII-HF G/GTNACC CutSmart < 10 10 < 10 100 37° No A Lambda
r t BstNI CC/WGG 3.1 10 100 100 75 60° No A Lambda a
t BstUI CG/CG CutSmart 50 100 25 100 60° No A Lambda u b
r t BstXI CCANNNNN/NTGG 3.1 < 10 50 100 25 37° 80° B Lambda I 3
r t BstYI R/GATCY 2.1 25 100 75 100 60° No A Lambda
r t e BstZ17I-HF GTA/TAC CutSmart 100 100 10 100 37° No A Lambda u
r t Bsu36I CC/TNAGG CutSmart 25 100 100 100 37° 80° C Lambda HindIII b
r t BtgI C/CRYGG CutSmart 50 100 100 100 37° 80° B pBR322
r BtgZI GCGATG(10/14) CutSmart 10 25 < 10 100 60° 80° A Lambda u 3, b, d
r t e Btsl-v2 GCAGTG(2/0) CutSmart 100 100 25 100 55° No A Lambda
r e BtsIMutI CAGTG(2/0) CutSmart 100 50 10 100 55° 80° A pUC19 b
r t BtsCI GGATG(2/0) CutSmart 10 100 25 100 50° 80° B Lambda
t Cac8I GCN/NGC CutSmart 50 75 100 100 37° 65° B Lambda u b
r t ClaI AT/CGAT CutSmart 10 50 50 100 37° 65° A Lambda dam- i u
r t @ CspCI (11/13)CAA(N)5GTGG(12/10) CutSmart + SAM 10 100 10 100 37° 65° A Lambda e
r t CviAII C/ATG CutSmart 50 50 10 100 25° 65° C Lambda
r CviKI-1 RG/CY CutSmart 25 100 100 100 37° No A pBR322 1, b
r t CviQI G/TAC 3.1 75 100* 100 75* 25° No C Lambda b
r t DdeI C/TNAG CutSmart 75 100 100 100 37° 65° B Lambda
r t DpnI GA/TC CutSmart 100 100 75 100 37° 80° B pBR322 u b
r t DpnII /GATC U 25 25 100* 25 37° 65° B Lambda dam- i
r t DraI TTT/AAA CutSmart 75 75 50 100 37° 65° A Lambda
r t e DraIII-HF CACNNN/GTG CutSmart < 10 50 10 100 37° No B Lambda u b
t DrdI GACNNNN/NNGTC CutSmart 25 50 10 100 37° 65° A pUC19 u 3
r EaeI Y/GGCCR CutSmart 10 50 < 10 100 37° 65° A Lambda I u b
r t EagI C/GGCCG 3.1 10 25 100 10 37° 65° B pXba u
r t e EagI-HF C/GGCCG CutSmart 25 100 100 100 37° 65° B pXba u
r t EarI CTCTTC(1/4) CutSmart 50 10 < 10 100 37° 65° B Lambda u b, d
r EciI GGCGGA(11/9) CutSmart 100 50 50 100 37° 65° A Lambda u 2
r t Eco53kI GAG/CTC CutSmart 100 100 < 10 100 37° 65° A pXba u 3, b
r t EcoNI CCTNN/NNNAGG CutSmart 50 100 75 100 37° 65° A Lambda b
r t EcoO109I RG/GNCCY CutSmart 50 100 50 100 37° 65° A Lambda HindIII I 3
r t @ EcoP15I CAGCAG(25/27) 3.1 + ATP 75 100 100 100 37° 65° A pUC19 e
r t EcoRI G/AATTC U 25 100* 50 50* 37° 65° C Lambda u
r t e EcoRI-HF G/AATTC CutSmart 10 100 < 10 100 37° 65° C Lambda u
r t EcoRV GAT/ATC 3.1 10 50 100 10 37° 80° A Lambda u
r t e EcoRV-HF GAT/ATC CutSmart 25 100 100 100 37° 65° B Lambda u
r t Esp3I CGTCTC(1/5) CutSmart 100 100 < 10 100 37° 65° B Lambda u
r FatI /CATG 2.1 10 100 50 50 55° 80° A pUC19
r FauI CCCGC(4/6) CutSmart 100 50 10 100 55° 65° A Lambda u 3, b, d
r t Fnu4HI GC/NGC CutSmart < 10 < 10 < 10 100 37° No A Lambda u a
r @ FokI GGATG(9/13) CutSmart 100 100 75 100 37° 65° A Lambda I u 3, b, d
r t FseI GGCCGG/CC CutSmart 100 75 < 10 100 37° 65° B pBC4 I u
r t FspI TGC/GCA CutSmart 10 100 10 100 37° No C Lambda u b
r FspEI CmC(12/16) CutSmart < 10 < 10 < 10 100 37° 80° B pBR322 I 1, e
Incubation Inactivation Supplied % Activity in NEBuffers Temp. Temp. Methylation
Enzyme Sequence NEBuffer 1.1 2.1 3.1 CutSmart (°C) (°C) Diluent Substrate Sensitivity Note(s)
r t HaeII RGCGC/Y CutSmart 25 100 10 100 37° 80° A Lambda u
r t HaeIII GG/CC CutSmart 50 100 25 100 37° 80° A Lambda
r HgaI GACGC(5/10) 1.1 100 100 25 100 37° 65° A phiX174 u 1
r t HhaI GCG/C CutSmart 25 100 100 100 37° 65° A Lambda u
r t HincII GTY/RAC 3.1 25 100 100 100 37° 65° B Lambda u
r HindIII A/AGCTT 2.1 25 100 50 50 37° 80° B Lambda 2
r t e HindIII-HF A/AGCTT CutSmart 10 100 10 100 37° 80° B Lambda
r t HinfI G/ANTC CutSmart 50 100 100 100 37° 80° A Lambda u
r t HinP1I G/CGC CutSmart 100 100 100 100 37° 65° A Lambda u
r HpaI GTT/AAC CutSmart < 10 75* 25 100 37° No A Lambda u 1
r t HpaII C/CGG CutSmart 100 50 < 10 100 37° 80° A Lambda u
r t HphI GGTGA(8/7) CutSmart 50 50 < 10 100 37° 65° B Lambda i u b, d
r Hpy99I CGWCG/ CutSmart 50 10 < 10 100 37° 65° A Lambda u
r t Hpy166II GTN/NAC CutSmart 100 100 50 100 37° 65° C pBR322 u
r Hpy188I TCN/GA CutSmart 25 100 50 100 37° 65° A pBR322 i 1, b
r Hpy188III TC/NNGA CutSmart 100 100 10 100 37° 65° B pUC19 i u 3, b
r t HpyAV CCTTC(6/5) CutSmart 100 100 25 100 37° 65° Lambda u 3, b, d
r HpyCH4III ACN/GT CutSmart 100 25 < 10 100 37° 65° A Lambda b
r t HpyCH4IV A/CGT CutSmart 100 50 25 100 37° 65° A pUC19 u
r t HpyCH4V TG/CA CutSmart 50 50 25 100 37° 65° A Lambda
r KasI G/GCGCC CutSmart 50 100 50 100 37° 65° B pBR322 u 3
r KpnI GGTAC/C 1.1 100 75 < 10 50 37° No A pXba 1
r t e KpnI-HF GGTAC/C CutSmart 100 25 < 10 100 37° No A pXba
r LpnPI CmCDG(10/14) CutSmart < 10 < 10 < 10 100 37° 65° B pBR322 1, e
r t MboI /GATC CutSmart 75 100 100 100 37° 65° A Lambda dam- i u
r @ MboII GAAGA(8/7) CutSmart 100* 100 50 100 37° 65° C Lambda dam- i b
r MfeI C/AATTG CutSmart 75 50 10 100 37° No A Lambda 2
r t e MfeI-HF C/AATTG CutSmart 75 25 < 10 100 37° No A Lambda
r t MluI A/CGCGT 3.1 10 50 100 25 37° 80° A Lambda u
r t e MluI-HF A/CGCGT CutSmart 25 100 100 100 37° No A Lambda u
r t MluCI /AATT CutSmart 100 10 10 100 37° No A Lambda
r t MlyI GAGTC(5/5) CutSmart 50 50 10 100 37° 65° A Lambda b, d
r t @ MmeI TCCRAC(20/18) CutSmart + SAM 50 100 50 100 37° 65° B phiX174 u b, c
r t MnlI CCTC(7/6) CutSmart 75 100 50 100 37° 65° B Lambda b
r MscI TGG/CCA CutSmart 25 100 100 100 37° 80° C Lambda I
r t MseI T/TAA CutSmart 75 100 75 100 37° 65° A Lambda
r t MslI CAYNN/NNRTG CutSmart 50 50 < 10 100 37° 80° A Lambda
r t MspI C/CGG CutSmart 75 100 50 100 37° No A Lambda
r t MspA1I CMG/CKG CutSmart 10 50 10 100 37° 65° B Lambda u
r MspJI mCNNR(9/13) CutSmart < 10 < 10 < 10 100 37° 65° B pBR322 1, e
r t MwoI GCNNNNN/NNGC CutSmart < 10 100 100 100 60° No B Lambda u
r @ NaeI GCC/GGC CutSmart 25 25 < 10 100 37° No A pXba u b
r @ NarI GG/CGCC CutSmart 100 100 10 100 37° 65° A pXba u
r Nb.BbvCI CCTCAGC(none/-2) CutSmart 25 100 100 100 37° 80° A pUB e
r Nb.BsmI GAATGC(none/-1) 3.1 < 10 50 100 10 65° 80° A pBR322 e
r Nb.BsrDI GCAATG(none/0) CutSmart 25 100 100 100 65° 80° A pUC19 e
r Nb.BssSI CACGAG (none/-1) 3.1 10 100 100 25 37° No B pUC19 e
r Nb.BtsI GCAGTG(none/0) CutSmart 75 100 75 100 37° 80° A phiX174 e
r t NciI CC/SGG CutSmart 100 25 10 100 37° No A Lambda u b
r t NcoI C/CATGG 3.1 100 100 100 100 37° 80° A Lambda
NcoI-HF C/CATGG CutSmart 50 100 10 100 37° 80° B Lambda
NdeI CA/TATG CutSmart 75 100 100 100 37° 65° A Lambda
r t @ NgoMIV G/CCGGC CutSmart 100 50 10 100 37° No A pXba u 1
r t NheI G/CTAGC 2.1 100 100 10 100 37° 65° C Lambda HindIII u
r t e NheI-HF G/CTAGC CutSmart 100 25 < 10 100 37° 80° C Lambda HindIII u
r t NlaIII CATG/ CutSmart < 10 < 10 < 10 100 37° 65° B phiX174
r NlaIV GGN/NCC CutSmart 10 10 10 100 37° 65° B pBR322 I u
r @ NmeAIII GCCGAG(21/19) CutSmart + SAM 10 10 < 10 100 37° 65° B phiX174 c
r t NotI GC/GGCCGC 3.1 < 10 50 100 25 37° 65° C pBC4 u
r t e NotI-HF GC/GGCCGC CutSmart 25 100 25 100 37° 65° A pBC4 u
r t NruI TCG/CGA 3.1 < 10 10 100 10 37° No A Lambda i u b
r t e NruI-HF TCG/CGA CutSmart 0 25 50 100 37° No A Lambda i u
r t NsiI ATGCA/T 3.1 10 75 100 25 37° 65° B Lambda
r t e NsiI-HF ATGCA/T CutSmart < 10 20 < 10 100 37° 80° B Lambda
r t NspI RCATG/Y CutSmart 100 100 < 10 100 37° 65° A Lambda
r Nt.AlwI GGATC(4/none) CutSmart 10 100 100 100 37° 80° A pUC101 dam-dcm- i e
r Nt.BbvCI CCTCAGC(-5/none) CutSmart 50 100 10 100 37° 80° A pUB u e
r Nt.BsmAI GTCTCN(1/none) CutSmart 100 50 10 100 37 65° A pBR322 u e
r Nt.BspQI GCTCTTC(1/none) 3.1 < 10 25 100 10 50° 80° B pUC19 e
r Nt.BstNBI GAGTC(4/none) 3.1 0 10 100 10 55° 80° A T7 e
r Nt.CviPII CCD(-3/none) CutSmart 10 100 25 100 37° 65° A pUC19 u e
r t PacI TTAAT/TAA CutSmart 100 75 10 100 37° 65° A pNEB193
r t PaeR7I C/TCGAG CutSmart 25 100 10 100 37° No A Lambda HindIII u
r PciI A/CATGT 3.1 50 75 100 50* 37° 80° B pXba
r t PflFI GACN/NNGTC CutSmart 25 100 25 100 37° 65° A pBC4 b
r t PflMI CCANNNN/NTGG 3.1 0 100 100 50 37° 65° A Lambda I 3, b, d
r @ PleI GAGTC(4/5) CutSmart 25 50 25 100 37° 65° A Lambda u b, d
r @ PluTI GGCGC/C CutSmart 100 25 < 10 100 37° 65° A pXba u b
r t PmeI GTTT/AAAC CutSmart < 10 50 10 100 37° 65° A Lambda u
r t PmlI CAC/GTG CutSmart 100 50 < 10 100 37° 65° A Lambda HindIII u
r t PpuMI RG/GWCCY CutSmart < 10 < 10 < 10 100 37° No B Lambda HindIII I
r t PshAI GACNN/NNGTC CutSmart 25 50 10 100 37° 65° A Lambda u
r PsiI TTA/TAA CutSmart 10 100 10 100 37° 65° B Lambda 3
r PspGI /CCWGG CutSmart 25 100 50 100 75° No A T7 I 3
r PspOMI G/GGCCC CutSmart 10 10 < 10 100 37° 65° B pXba I u
r PspXI VC/TCGAGB CutSmart < 10 100 25 100 37° No B Lambda HindIII u
r t PstI CTGCA/G 3.1 75 75 100 50* 37° 80° C Lambda
r t e PstI-HF CTGCA/G CutSmart 10 75 50 100 37° No C Lambda
r t PvuI CGAT/CG 3.1 < 10 25 100 < 10 37° No B pXba u
r t e PvuI-HF CGAT/CG CutSmart 25 100 100 100 37° No B pXba u
r t PvuII CAG/CTG 3.1 50 100 100 100* 37° No B Lambda
r t e PvuII-HF CAG/CTG CutSmart < 10 < 10 < 10 100 37° No B Lambda
r t RsaI GT/AC CutSmart 25 50 < 10 100 37° No A Lambda u
r @ RsrII CG/GWCCG CutSmart 25 75 10 100 37° 65° C Lambda u
r t SacI GAGCT/C 1.1 100 50 10 100 37° 65° A Lambda HindIII
r t e SacI-HF GAGCT/C CutSmart 10 50 < 10 100 37° 65° A Lambda HindIII u
r t @ SacII CCGC/GG CutSmart 10 100 10 100 37° 65° A pXba u
r t SalI G/TCGAC 3.1 < 10 < 10 100 < 10 37° 65° A Lambda HindIII u
r t e SalI-HF G/TCGAC CutSmart 10 100 100 100 37° 65° A Lambda HindIII u
r t SapI GCTCTTC(1/4) CutSmart 75 50 < 10 100 37° 65° B Lambda
r Sau3AI /GATC 1.1 100 50 10 100 37° 65° A Lambda u b
r Sau96I G/GNCC CutSmart 50 100 100 100 37° 65° A Lambda I u
r t SbfI CCTGCA/GG CutSmart 50 25 < 10 100 37° 80° A Lambda 3
r t e SbfI-HF CCTGCA/GG CutSmart 50 25 < 10 100 37° 80° B Lambda
r t e ScaI-HF AGT/ACT CutSmart 100 100 10 100 37° 80° B Lambda
r ScrFI CC/NGG CutSmart 100 100 100 100 37° 65° C Lambda I u 2, a
r SexAI A/CCWGGT CutSmart 100 75 50 100 37° 65° A pBC4 dcm- I 3, b, d
r SfaNI GCATC(5/9) 3.1 < 10 75 100 25 37° 65° B phiX174 u 3, b
r SfcI C/TRYAG CutSmart 75 50 25 100 37° 65° B Lambda 3
r t @ SfiI GGCCNNNN/NGGCC CutSmart 25 100 50 100 50° No C pXba I u
r t SfoI GGC/GCC CutSmart 50 100 100 100 37° No B Lambda HindIII I u
r @ SgrAI CR/CCGGYG CutSmart 100 100 10 100 37° 65° A Lambda u 1
r t SmaI CCC/GGG CutSmart < 10 < 10 < 10 100 25° 65° B Lambda HindIII u b
r SmlI C/TYRAG CutSmart 25 75 25 100 55° No A Lambda b
r SnaBI TAC/GTA CutSmart 50 50 10 100 37° 80° A T7 u 1
r t SpeI A/CTAGT CutSmart 75 100 25 100 37° 80° C Adenovirus-2
r t e SpeI-HF A/CTAGT CutSmart 25 50 10 100 37° 80° C pXba
r SphI GCATG/C 2.1 100 100 50 100 37° 65° B Lambda 2
r t e SphI-HF GCATG/C CutSmart 50 25 10 100 37° 65° B Lambda
r t e SrfI GCCC/GGGC CutSmart 10 50 0 100 37° 65° B pNEB193-SrFI u
r t SspI AAT/ATT U 50 100 50 50 37° 65° C Lambda
r t e SspI-HF AAT/ATT CutSmart 25 100 < 10 100 37° 65° B Lambda
r t StuI AGG/CCT CutSmart 50 100 50 100 37° No A Lambda I
r t StyD4I /CCNGG CutSmart 10 100 100 100 37° 65° B Lambda I u
r t StyI C/CWWGG 3.1 10 25 100 10 37° 65° A Lambda b
r t e StyI-HF C/CWWGG CutSmart 25 100 25 100 37° 65° A Lambda
r t SwaI ATTT/AAAT 3.1 10 10 100 10 25° 65° B pXba b, d
r t TaqaI T/CGA CutSmart 50 75 100 100 65° 80° B Lambda i
r t TfiI G/AWTC CutSmart 50 100 100 100 65° No C Lambda u
t TseI G/CWGC CutSmart 75 100 100 100 65° No B Lambda u 3
Tsp45I /GTSAC CutSmart 100 50 < 10 100 65° No A Lambda
t TspMI C/CCGGG CutSmart 50* 75* 50* 100 75° No B pUCAdeno u d
r t TspRI NNCASTGNN/ CutSmart 25 50 25 100 65° No B Lambda
r t Tth111I GACN/NNGTC CutSmart 25 100 25 100 65° No B pBC4 b
r t XbaI T/CTAGA CutSmart < 10 100 75 100 37° 65° A Lambda HindIII dam- i
r XcmI CCANNNNN/NNNNTGG 2.1 10 100 25 100 37° 65° C Lambda 2
r t XhoI C/TCGAG CutSmart 75 100 100 100 37° 65° A Lambda HindIII b
r t XmaI C/CCGGG CutSmart 25 50 < 10 100 37° 65° A pXba u 3
r t XmnI GAANN/NNTTC CutSmart 50 75 < 10 100 37° 65° A Lambda b
r ZraI GAC/GTC CutSmart 100 25 10 100 37° 80° B Lambda u
Activity of DNA Modifying Enzymes in CutSmart BufferA selection of DNA modifying enzymes were assayed in CutSmart Buffer, in lieu of their supplied buffers. Functional activity was compared to the activity in its supplied buffer, plus required supplements. Reactions were set up according to the recommended reaction conditions, with CutSmart Buffer replacing the supplied buffer.
Activity RequiredEnzyme in CutSmart Supplements
+ + + full functional activity + + 50–100% functional activity
+ 0–50% functional activity
Alkaline Phosphatase (CIP) + + +Antarctic Phosphatase + + + Requires Zn2+
Bst DNA Polymerase + + +CpG Methyltransferase (M. SssI) + + +DNA Polymerase I + + +DNA Polymerase I, Large (Klenow) Fragment + + +DNA Polymerase Klenow Exo– + + +DNase I (RNase-free) + + + Requires Ca2+
E. coli DNA Ligase + + + Requires NADEndonuclease III (Nth), recombinant + + +Endonuclease VIII + + +Exonuclease I + + +Exonuclease III + + +Exonuclease VII + + +Exonuclease V (Rec BCD) + + + Requires ATPGpC Methyltransferase (M. CviPI) + Requires DTTLambda Exonuclease + + McrBC + + +Micrococcal Nuclease + + + Requires Ca2+
Nuclease Bal-31 + + +phi29 DNA Polymerase + + +Quick Dephosphorylation Kit + + +RecJf + + +Shrimp Alkaline Phosphatase (rSAP) + + +T3 DNA Ligase + + + Requires ATP + PEGT4 DNA Ligase + + + Requires ATPT4 DNA Polymerase + + +T4 Phage β-glucosyltransferase (T4-BGT) + + +T4 Polynucleotide Kinase + + + Requires ATP + DTTT4 PNK (3´ phosphatase minus) + + + Requires ATP + DTTT5 Exonuclease + + +T7 DNA Ligase + + + Requires ATP + PEGT7 DNA Polymerase (unmodified) + + +T7 Exonuclease + + +Thermolabile ExoI + + +USER® Enzyme, recombinant + + +
Incubation Inactivation Supplied % Activity in NEBuffers Temp. Temp. Methylation
Enzyme Sequence NEBuffer 1.1 2.1 3.1 CutSmart (°C) (°C) Diluent Substrate Sensitivity Note(s)
Cut Smarter with NEB Restriction Enzymes
• > 215 restriction enzymes are supplied with a single buffer, CutSmart® Buffer
• The largest selection of enzyme specificities
• More recombinant enzymes than any other supplier
• 195 Time-Saver™ qualified enzymes digest DNA in 5-15 minutes and can also be safely used overnight with no loss of sample
• High Fidelity (HF®) enzymes have been engineered for reduced star activity and are 100% active in CutSmart Buffer
• Purple loading dye is included with most restriction enzymes, including all HF enzymes, for sharper bands and no UV shadow
Chart LegendU Supplied with its own unique reaction buffer that is
different from the four standard NEBuffers. Compatibility with the four standard NEBuffers is indicated by the chart.
SAM Supplied with a separate vial of S-adenosylmethionine (SAM). To obtain 100% activity, SAM should be added to the 1X reaction mix as specified on the product data card.
r Recombinant
t Time-Saver qualified
e Engineered for maximum performance
i dam methylation sensitivity
I dcm methylation sensitivity
u CpG methylation sensitivity (applies to eukaryotic genomic DNA only)
@ Indicates that the restriction enzyme requires two or more sites for cleavage
NEBuffer Compositions (1X)
NEBuffer 1.1
NEBuffer 2.1
NEBuffer 3.1
CutSmart Buffer
Buffers should be thawed completely and mixed thoroughly before using.
Note: The values listed in this table are approximate. They were obtained using each enzyme’s specific unit assay substrate DNA.
Updates to this chart can be found at NEBCutSmart.com.
Notesa. Ligation is < 10%b. Ligation is 25% – 75%c. Recutting after ligation is < 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 the recognition sequence contains variable sequences.
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 under any conditions
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