Compatible Cohesive Ends and Generation of New Restriction Sites

New restriction sites can be generated by ligation of DNA fragments with compatible cohesive or blunt ends. These new restriction sites may be generated by:

  1. Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
  2. Cleavage with two restriction endonucleases that produce blunt ends.
  3. Cleavage with two restriction endonucleases that produce compatible overhangs.

Restriction endonucleases that produce compatible cohesive ends often produce recleavable ligation products. The combinations listed were identified by computer analysis, and although we have tried to ensure their accuracy, they have not necessarily been confirmed by experimentation.

Where isoschizomers exist, only one member of each set is listed. Only enzymes available from New England Biolabs have been listed.

Enzymes that have degenerate recognition sequences (i.e. recognize more than one sequence) are followed by a specific sequence in parentheses and are only listed if a non-degenerate equivalent does not exist. Be aware that these degenerate enzymes will cleave sequences in addition to the one listed.

A "--" denotes a ligation product that cannot be recleaved.

| A | B | C | D | E | H | K | M | N | P | R | S | T | X |    single letter code

Enzyme Ligated To Recleaved By
Acc65I
(G/GTACC)
BanI (G/GTACC) Acc65I, BanI, KpnI, NlaIV, RsaI 
BsiWI, BsrGI RsaI
AccI
(GT/CGAC) AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI --
(GT/CGAC) ClaI, BstBI, TaqαI TaqαI
AciI
(C/CGC)
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqαI --
BsaHI (GR/CGCC), HinP1I, NarI AciI
HpaII HpaII
AclI
(AA/CGTT)
AccI (GT/CGAC), AciI, ClaI, BstBI, HinP1I, HpaII, NarI, TaqαI --
AgeI *+
(A/CCGGT)
AvaI (C/CCGGG), XmaI HpaII, NciI, ScrFI
BsaWI, BspEI BsaWI, HpaII
BsrFI (A/CCGGT), SgrAI (CA/CCGGTG) AgeI, BsaWI, BsrFI, HpaII
NgoMIV BsrFI, HpaII
ApaI
(GGGCC/C)
BanII (GGGCC/C), Bsp1286I (GGGCC/C) ApaI, BanII, Bsp120I, Bsp1286I, HaeIII, NlaIV, Sau96I
ApaLI
(G/TGCAC)
SfcI (C/TGCAG) BsgI
ApoI
(A/AATTY) EcoRI ApoI, Tsp509I
(G/AATTY) EcoRI ApoI, EcoRI, Tsp509I
(R/AATTY) MfeI, Tsp509I Tsp509I
AscI
(GG/CGCGCC)
AflIII (A/CGCGT), MluI BstUI, HhaI
BssHII BssHII, BstUI, Cac8I, HhaI
AseI +
(AT/TAAT)
BfaI, Csp6I, NdeI --
MseI MseI
AsiSI
(GCGAT/CGC)
BsiEI (CGAT/CG) DpnII, PvuI
PacI MseI
PvuI DpnII, PvuI
AvaI
(C/CCGGG) AgeI, BsaWI, BspEI, BsrFI (R/CCGGY), NgoMIV, SgrAI (CR/CCGGYG) HpaII, NciI, ScrFI
(C/TCGAG) XhoI AvaI, TaqαI, XhoI
(C/TCGAG) SalI TaqαI
(C/CCGGG) XmaI AvaI, BsaJI, HpaII, NciI, ScrFI, SmaI
AvaII
(G/GWCC)
PpuMI (RG/GACCY) AvaII, NlaIV, Sau96I
RsrII AvaII, Sau96I
PpuMI (RG/GTCCY) AvaII, BsmFI, NlaIV, Sau96I
AvrII
(C/CTAGG)
NheI, SpeI, XbaI BfaI
StyI (C/CTAGG) AvrII, BfaI, BsaJI, StyI
BamHI *
(G/GATCC)
BclI, DpnII AlwI, DpnII
BglII, BstYI (R/GATCY) AlwI, BstYI, DpnII
BstYI (G/GATCC) AlwI, BamHI, BstYI, DpnII, NlaIV
BanI
(G/GTACC) Acc65I Acc65I, BanI, KpnI, NlaIV, RsaI
(G/GCGCC) KasI BanI, BsaHI, HaeII, HhaI, KasI, NarI, NlaIV
(G/GTACC) BsiWI, BsrGI RsaI
BanII
(GGGCC/C) ApaI, Bsp1286I (GGGCC/C) ApaI, BanII, Bsp1286I, HaeIII, NlaIV, Sau96I
(GAGCT/C) Bsp1286I (GAGCT/C), SacI AluI, BanII, BsiHKAI, Bsp1286I, SacI
BclI
(T/GATCA)
BamHI, BstYI (R/GATCY) AlwI, DpnII
BglII, MboI DpnII
BfaI
(C/TAG)
AseI, Csp6I, MseI, NdeI --
BglII
(A/GATCT)
BamHI, BstYI (R/GATCY) AlwI, BstYI, DpnII
BclI, DpnII DpnII
BsaHI
(GR/CGYC) AccI (GT/CGAC), ClaI, BstBI, TaqαI --
(GA/CGYC) AciI, HinP1I HgaI
(GG/CGYC) AciI, HinP1I HhaI
(GG/CGYC) HpaII AciI
(GA/CGYC) NarI BsaHI, HgaI
(GG/CGYC) NarI BanI, BsaHI, HaeII, HhaI, NarI, NlaIV
BsaWI
(W/CCGGW)
AgeI, BsrFI (R/CCGGY), SgrAI (CR/CCGGYG) AgeI, BsaWI, BsrFI, HpaII
AvaI (C/CCGGG), XmaI HpaII, NciI, ScrFI
BspEI BsaWI, BspEI, HpaII
BsrFI (R/CCGGY), NgoMIV BsrFI, HpaII
NgoMIV HpaII
BsiEI
(CGAT/CG) PacI MseI
(CGAT/CG) PvuI BsiEI, DpnII, PvuI
(CGGC/CG) SacII AciI
BsiHKAI
(GTGCA/C)
Bsp1286I (GTGCA/C) BsiHKAI, Bsp1286I
Bsp1286I (GAGCA/C) BsiHKAI, Bsp1286I
Bsp1286I (GAGCT/C), SacI AluI, BanII, BsiHKAI, Bsp1286I, SacI
NsiI --
PstI, SbfI BsgI
BsiWI
(C/GTACG)
Acc65I, BanI (G/GTACC), BsrGI RsaI
Bsp1286I
(GGGCC/C) ApaI, BanII (GGGCC/C) ApaI, BanII, Bsp1286I, HaeIII, NlaIV, Sau96I
(GTGCA/C) BsiHKAI ApaLI, BsiHKAI, Bsp1286I
(GGGCC/C) BanII (GGGCC/C) BanII, Bsp1286I
(GAGCT/C) BanII (GAGCT/C), BsiHKAI, SacI AluI, BanII, BsiHKAI, Bsp1286I, SacI
(GWGCW/C) BsiHKAI BsiHKAI, Bsp1286I
(GTGCA/C) NsiI --
(GTGCA/C) PstI, SbfI BsgI
BspEI
(T/CCGGA)
AgeI, BsaWI, BsrFI (R/CCGGY), SgrAI (CR/CCGGYG) BsaWI, HpaII
AvaI (C/CCGGG), XmaI HpaII, NciI, ScrFI
BsaWI BsaWI, BspEI, HpaII
BsrFI (R/CCGGY), NgoMIV HpaII
BspHI
(T/CATGA)
FatI, NcoI, PciI FatI, NlaIII
BsrFI
(A/CCGGY) AgeI, BsaWI AgeI, BsaWI, BsrFI, HpaII
(G/CCGGY) AgeI, BsaWI, NgoMIV BsrFI, HpaII
(R/CCGGY) AvaI (C/CCGGG), XmaI HpaII, NciI, ScrFI
(A/CCGGY) BsaWI, BspEI BsaWI, HpaII
(R/CCGGY) BsaWI, BspEI HpaII
(G/CCGGY) NgoMIV BsrFI, Cac8I, HpaII, NaeI
(CR/CCGGYG) SgrAI BsrFI, HpaII
BsrGI
(T/GTACA)
Acc65I, BanI (G/GTACC), BsiWI RsaI
BssHII
(G/CGCGC)
MluI BstUI, HhaI
AscI BssHII, BstUI, Cac8I, HhaI
BstBI
(TT/CGAA)
AccI (GT/CGAC), ClaI, TaqI TaqI
AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI --
BstYI
(A/GATCY) BamHI, BglII AlwI, BstYI, DpnII
(G/GATCY) BamHI AlwI, BamHI, BstYI, DpnII, NlaIV
(R/GATCY) BclI, DpnII DpnII
(G/GATCY) BclI, DpnII AlwI, DpnII
(A/GATCY) BglII BglII, BstYI, DpnII
ClaI
(AT/CGAT)
AccI (GT/CGAC), BstBI, TaqI TaqI
AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI --
DpnII/MboI/
Sau3AI (/GATC)
BamHI, BstYI (R/GATCC) AlwI, DpnII
BclI, BglII, BstYI (R/GATCY) DpnII
EaeI
(Y/GGCCR) PspOMI HaeIII, Sau96I
(C/GGCCR) EagI BsiEI, EaeI, EagI, HaeIII
(T/GGCCR) EagI EaeI, HaeIII
(C/GGCCR) NotI AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII
(T/GGCCR) NotI AciI, EaeI, Fnu4HI, HaeIII
EagI *
(C/GGCCG)
PspOMI HaeIII, Sau96I
EaeI (Y/GGCCR) EaeI, HaeIII
EaeI (C/GGCCG) BsiEI, EaeI, EagI, HaeIII
NotI AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII
EcoRI *+
(G/AATTC)
ApoI (G/AATTC) ApoI, EcoRI, Tsp509I
ApoI (R/AATTY) ApoI, Tsp509I
MfeI, Tsp509I Tsp509I
FatI
(/CATG)
BspHI, NcoI, PciI FatI, NlaIII
HinP1I
(G/CGC)
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI --
AciI, BsaHI (GR/CGCC), NarI HhaI
BsaHI (GR/CGTC) HgaI
HpaII AciI
HpaII/MspI
(C/CGG)
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI --
AciI, BsaHI (GR/CGCC), HinP1I, NarI AciI
KasI
(G/GCGCC)
BanI (G/GCGCC) BanI, BsaHI, HaeII, HhaI, KasI, NarI, NlaIV
MfeI *+
(C/AATTG)
ApoI (R/ATTTY), EcoRI, Tsp509I Tsp509I
MluI
(A/CGCGT)
AscI, BssHII BstUI, HhaI
MseI
(T/TAA)
AseI MseI
BfaI, Csp6I, NdeI --
NarI
(GG/CGCC)
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqαI --
AciI, HinP1I HhaI
BsaHI (GR/CGCC) BanI, BsaHI, HaeII, HhaI, NarI, NlaIV
BsaHI (GR/CGTC) BsaHI, HgaI
HpaII AciI
NcoI *+
(C/CATGG)
BspHI, FatI, PciI FatI, NlaIII
NdeI
(CA/TATG)
AseI, BfaI, Csp6I, MseI --
NgoMIV
(G/CCGGC)
AgeI, BsaWI, BsrFI (R/CCGGY), SgrAI BsrFI, HpaII
AvaI (C/CCGGG), XmaI HpaII, NciI, ScrFI
BsaWI, BspEI HpaII
BsrFI (R/CCGGC), SgrAI BsrFI, Cac8I, HpaII, NaeI
NheI *+
(G/CTAGC)
AvrII, SpeI, StyI (C/CTAGG), XbaI BfaI
NlaIII
(CATG/)
SphI, NspI NlaIII
NotI *+
(GC/GGCCGC)
PspOMI AciI, EaeI, Fnu4HI, HaeIII
EagI AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII
EaeI (Y/GGCCR) AciI, BsiEI, EaeI, Fnu4HI, HaeIII
NsiI
(ATGCA/T)
BsiHKAI (GTGCA/C), Bsp1286I (GTGCA/C), PstI, SbfI --
NspI
(RCATG/Y)
NlaIII, SphI NlaIII
PacI +
(TTAAT/TAA)
AsiSI, BsiEI (CGAT/CG), PvuI MseI
PciI
(A/CATGT)
BspHI, FatI, NcoI FatI, NlaIII
PpuMI
(RG/GWCCY) AvaII, RsrII AvaII, Sau96I
(GG/GTCCY) AvaII, RsrII AvaII, BsmFI, NlaIV, Sau96I
(GG/GACCY) AvaII, RsrII AvaII, NlaIV, Sau96I
PspOMI
(G/GGCCC)
EaeI (Y/GGCCR), EagI HaeIII, Sau96I
NotI AciI, Fnu4HI, HaeIII, Sau96I
PspXI
(VC/TCGAGB)
XhoI, TliI XhoI, TliI
SalI TaqαI
PstI *
(CTGCA/G)
BsiHKAI, Bsp1286I (GTGCA/C) BsgI
NsiI --
SbfI PstI
PvuI *
(CGAT/CG)
AsiSI DpnI, PvuI
PacI MseI
BsiEI (CGAT/CG) BsiEI, DpnII, PvuI
RsrII
(CG/GWCCG)
AvaII, PpuMI (RG/GACCY) AvaII, Sau96I
PpuMI (RG/GACCY) AvaII, NlaIV, Sau96I
PpuMI (RG/GTCCY) AvaII, BsmFI, NlaIV, Sau96I
SacI *
(GAGCT/C)
BanII (GAGCT/C), BsiHKAI, Bsp1286I (GAGCT/C) AluI, BanII, BsiHKAI, Bsp1286I, SacI
SacII
(CCGC/GG)
BsiEI (CGGC/CG) AciI
SalI *+
(G/TCGAC)
PspXI, XhoI TaqαI
SbfI *
(CCTGCA/GG)
BsiHKAI, Bsp1286I (GTGCA/C) BsgI
NsiI --
PstI PstI
SfcI
(C/TGCAG)
ApaLI BsgI
SgrAI
(CR/CCGGYG)
See BsrFI  
SpeI *+
(A/CTAGT)
AvrII, NheI, StyI (C/CTAGG), XbaI BfaI
SphI *
(GCATG/C)
NlaIII, NspI NlaIII
StyI * AvrII AvrII, BfaI, BsaJI, StyI
(C/CTAGG) NheI, SpeI, XbaI BfaI
(C/CATGG) BspHI NlaIII
NcoI BsaJI, NcoI, NlaIII, StyI
TaqαI
(T/CGA)
AccI (GT/CGAC), ClaI, BstBI TaqαI
AciI, AclI, BsaHI (GR/CGYC), HinP1I,HpaII, NarI --
Tsp509I
(/AATT)
ApoI (R/AATTY), EcoRI, MfeI Tsp509I
XbaI +
(T/CTAGA)
AvrII, NheI, SpeI, StyI (C/CTAGG) BfaI
XhoI+/TliI
(C/TCGAG)
PspXI XhoI, TliI
SalI TaqαI
XmaI
(C/CCGGG)
AgeI, BsaWI, BspEI, BsrFI, NgoMIV, SgrAI HpaII, NciI, ScrFI
AvaI (C/CCGGG) AvaI, BsaJI, HpaII, NciI, ScrFI, SmaI, XmaI


* HF (high fidelity) versions of these enzymes are available for simplified reactions and reduced star activity, at no additional cost.

+ A RE-Mix version of this, or the HF version of this enzyme is available.