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  • Heat Inactivation

    Heat inactivation is a convenient method for stopping a restriction endonuclease reaction. Incubation at 65°C for 20 minutes inactivates the majority of restriction endonucleases that have an optimal incubation temperature of 37°C. Enzymes that cannot be inactivated at 65°C can often be inactivated by incubation at 80°C for 20 minutes. The table below indicates whether or not an enzyme can be heat inactivated and the temperature needed to do so.

    Heat inactivation was performed as follows to approximate a typical experiment. A 50 µl reaction mixture containing the appropriate NEBuffer, 0.5 µg of calf thymus DNA, and 5 or 10 µl of restriction endonuclease (at selling concentration) was incubated at 37°C for 60 minutes and then at 65°C or 80°C for 20 minutes. 0.5 µg of substrate DNA (usually lambda) was added to the reaction mixture and incubated at the optimal reaction temperature of the enzyme for 60 minutes. Any digestion (complete or partial) of the substrate DNA after the second incubation, as seen by agarose gel electrophoresis, was interpreted as incomplete heat inactivation.

    Please also check these additional information about:
    NEBuffer Activity | Activity at 37°C | Time-Saver Enzymes

    EnzymeHeat Inactivation TemperatureHeat Inactivation Time
    AatII80°C20 min
    AbaSI65°C20 min
    Acc65I65°C20 min
    AccI80°C20 min
    AciI65°C20 min
    AclINo--
    AcuI65°C20 min
    AfeI65°C20 min
    AflII65°C20 min
    AflIII80°C20 min
    AgeI65°C20 min
    AgeI-HF65°C20 min
    AhdI65°C20 min
    AleI80°C20 min
    AluI80°C20 min
    AlwINo--
    AlwNI80°C20 min
    ApaI65°C20 min
    ApaLINo--
    ApeKINo--
    ApoI80°C20 min
    AscI80°C20 min
    AseI65°C20 min
    AsiSI80°C20 min
    AvaI80°C20 min
    AvaII80°C20 min
    AvrIINo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    BaeGI80°C20 min
    BaeI65°C20 min
    BamHINo--
    BamHI-HF®No--
    BanI65°C20 min
    BanII80°C20 min
    BbsI65°C20 min
    BbvCINo--
    BbvI65°C20 min
    BccI65°C20 min
    BceAI65°C20 min
    BcgI65°C20 min
    BciVI80°C20 min
    BclINo--
    BcoDINo--
    BfaI80°C20 min
    BfuAI65°C20 min
    BfuCI80°C20 min
    BglI65°C20 min
    BglIINo--
    BlpINo--
    BmgBI65°C20 min
    BmrI65°C20 min
    BmtI65°C20 min
    BmtI-HF®65°C20 min
    BpmI65°C20 min
    Bpu10I80°C20 min
    BpuEI65°C20 min
    BsaAINo--
    BsaBI80°C20 min
    BsaHI80°C20 min
    BsaI65°C20 min
    BsaI-HF®65°C20 min
    BsaJI80°C20 min
    BsaWI80°C20 min
    BsaXINo--
    BseRI80°C20 min
    BseYI80°C20 min
    BsgI65°C20 min
    BsiEI80°C20 min
    BsiHKAINo--
    BsiWI65°C20 min
    BslINo--
    BsmAINo--
    BsmBI80°C20 min
    BsmFI80°C20 min
    BsmI80°C20 min
    BsoBI80°C20 min
    Bsp1286I65°C20 min
    BspCNI80°C20 min
    BspDI80°C20 min
    BspEI80°C20 min
    BspHI80°C20 min
    BspMI65°C20 min
    BspQI80°C20 min
    BsrBI80°C20 min
    BsrDI80°C20 min
    BsrFINo--
    BsrGI80°C20 min
    BsrI80°C20 min
    BssHII65°C20 min
    BssKI80°C20 min
    BssSI80°C20 min
    BstAPI80°C20 min
    BstBINo--
    BstEII No--
    BstEII-HF®No--
    BstNINo--
    BstUINo--
    BstXI80°C20 min
    BstYINo--
    BstZ17INo--
    Bsu36I80°C20 min
    BtgI80°C20 min
    BtgZI80°C20 min
    BtsCI80°C20 min
    BtsI80°C20 min
    BtsIMutI80°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    Cac8I65°C20 min
    ClaI65°C20 min
    CspCI65°C20 min
    CviAII65°C20 min
    CviKI-1No--
    CviQINo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    DdeI65°C20 min
    DpnI80°C20 min
    DpnII65°C20 min
    DraI65°C20 min
    DraIII65°C20 min
    DraIII-HF®No--
    DrdI65°C20 min
    DvuIIINo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    EaeI65°C20 min
    EagI65°C20 min
    EagI-HF®65°C20 min
    EarI65°C20 min
    EciI65°C20 min
    Eco53kI65°C20 min
    EcoNI65°C20 min
    EcoO109I65°C20 min
    EcoP15I65°C20 min
    EcoRI65°C20 min
    EcoRI-HF®65°C20 min
    EcoRV80°C20 min
    EcoRV-HF®65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    FatI80°C20 min
    FauI65°C20 min
    Fnu4HINo--
    FokI65°C20 min
    FseI65°C20 min
    FspEI80°C20 min
    FspINo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    HaeII80°C20 min
    HaeIII80°C20 min
    HgaI65°C20 min
    HhaI65°C20 min
    HincII65°C20 min
    HindIII80°C20 min
    HindIII-HF®80°C20 min
    HinfI80°C20 min
    HinP1I65°C20 min
    HpaINo--
    HpaII80°C20 min
    HphI65°C20 min
    Hpy166II65°C20 min
    Hpy188I65°C20 min
    Hpy188III65°C20 min
    Hpy99I65°C20 min
    HpyAV65°C20 min
    HpyCH4III65°C20 min
    HpyCH4IV65°C20 min
    HpyCH4V65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    I-CeuI65°C20 min
    I-SceI65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    KasI65°C20 min
    KpnINo--
    KpnI-HF®No--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    LpnPI65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    MboI65°C20 min
    MboII65°C20 min
    MfeINo--
    MfeI-HF®No--
    MluCINo--
    MluI80°C20 min
    MlyI65°C20 min
    MmeI65°C20 min
    MnlI65°C20 min
    MscI80°C20 min
    MseI65°C20 min
    MslI80°C20 min
    MspA1I65°C20 min
    MspINo--
    MspJI65°C20 min
    MwoINo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    NaeINo--
    NarI65°C20 min
    Nb.BbvCI80°C20 min
    Nb.BsmI80°C20 min
    Nb.BsrDI80°C20 min
    Nb.BtsI80°C20 min
    NciINo--
    NcoI80°C20 min
    NcoI-HF®80°C20 min
    NdeI65°C20 min
    NgoMIVNo--
    NheI65°C20 min
    NheI-HF®80°C20 min
    NlaIII65°C20 min
    NlaIV65°C20 min
    NmeAIII65°C20 min
    NotI65°C20 min
    NotI-HF®65°C20 min
    NruINo--
    NsiI65°C20 min
    NspI65°C20 min
    Nt.AlwI80°C20 min
    Nt.BbvCI80°C20 min
    Nt.BsmAI65°C20 min
    Nt.BspQI80°C20 min
    Nt.BstNBI80°C20 min
    Nt.CviPII65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    PacI65°C20 min
    PaeR7INo--
    PciI80°C20 min
    PflFI65°C20 min
    PflMI65°C20 min
    PhoINo--
    PI-PspINo--
    PI-SceI65°C20 min
    PleI65°C20 min
    PluTI65°C20 min
    PmeI65°C20 min
    PmlI65°C20 min
    PpuMINo--
    PshAI65°C20 min
    PsiI65°C20 min
    PspGINo--
    PspOMI65°C20 min
    PspXINo--
    PstI80°C20 min
    PstI-HF®No--
    PvuINo--
    PvuI-HF®No--
    PvuIINo--
    PvuII-HF®No--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    RsaINo--
    RsrII65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    SacI65°C20 min
    SacI-HF®65°C20 min
    SacII65°C20 min
    SalI65°C20 min
    SalI-HF®65°C20 min
    SapI65°C20 min
    Sau3AI65°C20 min
    Sau96I65°C20 min
    SbfI80°C20 min
    SbfI-HF®80°C20 min
    ScaI80°C20 min
    ScaI-HF®80°C20 min
    ScrFI65°C20 min
    SexAI65°C20 min
    SfaNI65°C20 min
    SfcI65°C20 min
    SfiINo--
    SfoINo--
    SgrAI65°C20 min
    SmaI65°C20 min
    SmlINo--
    SnaBI80°C20 min
    SpeI80°C20 min
    SpeI-HF®80°C20 min
    SphI-HF®65°C20 min
    SphI65°C20 min
    SspI65°C20 min
    SspI-HF®No--
    StuINo--
    StyD4I65°C20 min
    StyI65°C20 min
    StyI-HF®65°C20 min
    SwaI65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    TaqαI80°C20 min
    TfiINo--
    TliINo--
    TseINo--
    Tsp45INo--
    Tsp509INo--
    TspMINo--
    TspRINo--
    Tth111INo--
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    XbaI65°C20 min
    XcmI65°C20 min
    XhoI65°C20 min
    XmaI65°C20 min
    XmnI65°C20 min
    EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
    ZraI80°C20 min