Compatible Cohesive Ends

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  • New restriction sites can be generated by ligation of DNA fragments with compatible ends. These ends 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.

    Compatible ends, generated by each of the above methods, can be ligated to produce DNA sequences that often contain useful restriction endonuclease sites. Generation of these sites is detailed in the following tables.

    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)
    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