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  • Activity of Restriction Enzymes in PCR Buffers

    Frequently, a PCR product must be further manipulated by cleavage with restriction enzymes. For convenience, restriction enzyme digestion can be performed directly in the PCR mix without any purification of the DNA. This table summarizes the percent activity of restriction enzymes on the DNA in the Taq, Phusion® or Q5® PCR mixes described below.

    In these reactions, 5 units of restriction enzyme were incubated at the appropriate reaction temperature for 1 hour in a PCR mix containing 1 µg of DNA and 1 unit of DNA Polymerase in a 50 µl reaction volume with a final buffer concentration of 1X, and supplemented with dNTPs (200 µM final concentration). Enzyme activity was analyzed by gel electrophoresis.

    Notes: The polymerase is still active and can alter the ends of DNA fragments after they have been cleaved, affecting subsequent ligation. Primers containing the recognition site of the restriction enzyme can act as competitive inhibitors in the cleavage reaction. The use of restriction enzymes under non-optimal conditions does increase the likelihood of star activity. If any problems are encountered, the DNA should be purified using a commercial spin column (we recommend the Monarch PCR & DNA Cleanup Kit, NEB# T1030) or by phenol/chloroform followed by alcohol precipitation.

    *It has been shown that the addition of 1x restriction enzyme buffer may help to improve the ability of some enzymes to cleave.

    Chart Legend: Cleavage in extension mix with 5 units of enzyme: 
    +++ complete cleavage;   ++ ~50% cleavage;   + ~25% cleavage; – no activity

    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    AatII<++<+++<++++
    Acc65I++++++<+<+<++
    AccI++<++<+<+++++++
    AciI++++++++++++++
    AclI++++++<+<+++++++
    AcuI++++++++<+++++++
    AfeI++++++++<+++++++
    AflII<++<+<++<+
    AflIII<+<+++++<+<+
    AgeI (1) (2)+++++++++++++<+
    AgeI-HF® (2)++++++++<+++++++
    AhdI+++<+--<+<+
    AleI++++++<+<+++<+
    AluI++++++++++++++++
    AlwI<++-<+<+<+<+
    AlwNI<++<++++++<++
    ApaI @25°C++++++<+<+<+-
    ApaLI++++++<+<+++++++
    ApeKI @75°C<++<+++++++<++
    ApoI @50°C (1)++++++++++++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    ApoI-HF+++++++++++++++
    AscI (2)++++++<+<+<+-
    AseI+++++<+++++
    AsiSI++++++++<+++++++
    AvaI+++++++++<+++++
    AvaII++++++++<+++++++
    AvrII++++++<+<+++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    BaeGI+++++++++<+++++++
    BaeI @25°C--++<+<+<+
    BamHI (1)+++++++++<+++++++
    BamHI-HF®++++++-<+<+++
    BanI+++++++++<+++++++
    BanII+++++++++<+++++++
    BbsI++++++<+<+++++++
    BbvCI<+++++--<+<+
    BbvI++++++++<+++++++
    BccI<+<+<+<+<+<+
    BceAI<+<+++<++<+
    BcgI+++<++
    BciVI----<+-
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    BclI @50°C+++++++++++++++++
    BcoDI<+<++<++<+
    BfaI<++--<+--
    BfuAI @50°C<++<+++-<+-
    BfuCI<+<+<+<+<+
    BglI<++<++++<+<+
    BglII<+<++++<+<+
    BlpI<++<++<+<+<+-
    BmgBI<+-+++<+<+
    BmrI<++<+++++<+++++++
    BmtI (1)++++++++<+++++++
    BmtI-HF®+++++<++++++
    BpmI<+<++++<+<++<++
    Bpu10I<+<++++<++++++
    BpuEI++++++++-<++<++
    BsaAI++++++++++++++++
    BsaBI @60°C<++++<++++++
    BsaHI++++++++++++++++
    BsaI (1)+++<+++-+
    BsaI-HF®++++<+<+<+
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    BsaJI @60°C++++++++<+++++++
    BsaWI @60°C<++<++++<+++
    BsaXI<++<++<+<+<+<+
    BseRI++++++++<++++
    BseYI++++++++++++++++
    BsgI<+<++<+<+<+
    BsiEI @60°C++++++++<+++++
    BsiHKAI @65°C<+-+++--
    BsiWI @55°C++++++++<+++++++
    BslI @55°C+++++++++++++++++
    BsmAI @55°C+++++++++++<+<+
    BsmBI @55°C<++<+++++<+<+
    BsmFI @65°C<+<++++++++
    BsmI @65°C++++++<++++++
    BsoBI+++++++++++++++++
    Bsp1286I+<+<+<+<+<+
    BspCNI @25°C<+<++<+--
    BspDI<++<++++<+++++++
    BspEI--<+<+--
    BspHI<+++++++<+++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    BspMI++++++<+<+<+
    BspQI @50°C<+++++++++++++
    BsrBI+++++++<+++++++
    BsrDI @65°C<+<++
    BsrFI<+<++++<++++
    BsrGI (1)<+<+++++<++++
    BsrI @65°C+++++++++<++++++
    BssHII @50°C++++++<+++++++
    BssKI @60°C++++++<+<+++++
    BssSαI+++++++++<+++++++
    BssSI+++++++++<++++++
    BstAPI @60°C<+
    BstBI @65°C+++++++++++++++++
    BstEII  @60°C (1) (2)++++++<+<+++++++
    BstEII-HF®++++<+<+++++
    BstNI @60°C++++++<+<+<+<+
    BstUI @60°C++++++<+<+++++
    BstXI<++<+++++<+
    BstYI @60°C++++++<+<++++
    BstZ17I+++++++++<+++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    BstZ17I-HF+++++++-++++++
    Bsu36I<+<+<+<+<++
    BtgI<+++++<+<+<+
    BtgZI @60°C+++++++++++++
    BtsαI @55°C-++++-++++++
    BtsCI @50°C++++++<+<+++++++
    BtsI @55°C++++++<+<+++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    Cac8I++++++<+<++++++
    ClaI+++++<+<+<+++
    CspCI<+<++-<+<+
    CviAII @25°C+++++++<+++++++
    CviKI-1-+++++<+++++++
    CviQI @25°C+++++++++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    DdeI+++++++++++++++
    DpnI<+++++++++++++
    DpnII++++++++++++++++
    DraI++++++++<+++++++
    DraIII (1)<+<+<+<+<+
    DraIII-HF®++++++++++++
    DrdI++++++++<+++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    EaeI++++++-<+<+<+
    EagI (1)<+<+++++++++++++
    EagI-HF®+++++<+++++
    EarI+++++++<++<+
    EciI<+<++++++<++<++
    Eco53kI<+
    EcoNI<++++++<+++++++
    EcoO109I++++++-<+<++
    EcoP15I<+<++<+<++
    EcoRI (1) (2)+++++<+--
    EcoRI-HF® (2)+++++++<+++++++
    EcoRV (1) (2)<+<++<+-<+
    EcoRV-HF® (2)++<+<++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    FatI @55°C+++++++<+<++++
    FauI @55°C++++++<++++++
    Fnu4HI++++++<+<++++
    FokI++++++++++++++
    FseI<++++<++++-
    FspI+<+++<+++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    HaeII+++++++++<+++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    HaeIII+++++++++<+++++++
    HgaI+++<++<+<++<++
    HhaI+++++++++<+++++++
    HincII++++++<+<+++++++
    HindIII (1)++++++<++++++
    HindIII-HF®++++<+<+++++++
    HinfI++++++++++++++++
    HinP1I++++++++++++++++
    HpaI+++++++++<+++++++
    HpaII++++<+<+<+<+
    HphI+++<++<+<+<+<+
    Hpy166II+++++++++++++
    Hpy188I+++++++<+++++
    Hpy188III<++<+<++<+
    Hpy99I+++++++-<+<+
    HpyAV++++++-+++
    HpyCH4III<++<+++<+<++<++
    HpyCH4IV+++++<+<+++++++
    HpyCH4V++++++<+<+++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    KasI-++++++<++++-
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    KpnI (1) (2)+++++++++++<+
    KpnI-HF® (2)++++++-<+<+
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    MboI+++++++++<+++++++
    MboII+++++++++++
    MfeI (1) (2)+++++<+<+++++
    MfeI-HF® (2)++--+++<+
    MluCI++++<+<++++
    MluI (1)+++++++++++++
    MluI-HF®++++++-++++
    MlyI<+++++++<++
    MmeI<+<+++-<+<+
    MnlI++++++++++
    MscI<+<++<+<+<+
    MseI+<+<+<+<+<+
    MslI+++++++<++++++
    MspA1I+++++++++<++++++
    MspI+++++++++<++++++
    MwoI @60°C+++++++++++++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    NaeI<+<++<+<+<+
    NarI--++<+++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    NciI++++++<+<++<+
    NcoI (1) (2)+++++++<+++++
    NcoI-HF® (2)++++++-<++++
    NdeI++<+++++++++<+
    NgoMIV--+<+<+<+
    NheI (1) (2)++++++<+<+++++++
    NheI-HF® (2)++++++-<+++++
    NlaIII+++<++<+++<+
    NlaIV<+++++++<+++++++
    NmeAIII<+<++++-<+<+
    NotI (1) (2)++++++<+<+<+
    NotI-HF® (2)+++++<+<+<++
    NruI (1)++++++++++
    NruI-HF®+++++--+-
    NsiI (1)+++++++++++++
    NsiI-HF®+++++++++++++++++
    NspI++++++<+<++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    PacI (2)++++++<+<++++++
    PaeR7I++++++<+<+++++++
    PciI+<+-<+--
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    PflFI++++++<+<+<++
    PflMI+++++<++++++
    PhoI @75°C<++++++++++++++++
    PleI++++<+<+<+<+
    PluTI<+
    PmeI++++++<+<+++++++
    PmlI+---+<+
    PpuMI+++++++++<+++++++
    PshAI++++++<+<+<+<+
    PsiI+++++<+<+<+
    PspGI @75°C++++++++++++++++++
    PspOMI+++++++<+++++++
    PspXI++++++++<+++++++
    PstI (1)++++++<+<+
    PstI-HF®+++++++<++++
    PvuI (1)<+<++++<+-<+
    PvuI-HF®+++++++++<++++++
    PvuII (1)+++++++<+++++++
    PvuII-HF®+++--<+<+
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    RsaI++++++++<+++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    RsrII<++<++--<+<+
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    SacI (1)+++++++<+++++
    SacI-HF®++++++<+<+<+++
    SacII+++++++++<++++
    SalI (1) (2)<+<++++--
    SalI-HF® (2)+++++++<+++++
    SapI<++<++++<+++++
    Sau3AI++++++<+<+<+<+
    Sau96I<++<++++++++++
    SbfI (1)<++<+++<+<++++
    SbfI-HF®++--<+<+
    ScaI (1) (2)<++<++++++++<+<+
    ScaI-HF® (2)++<+<+--
    ScrFI++++++++++++++++++
    SexAI+++++++++<+++++++
    SfaNI--++<+<++<++
    SfcI++++++<+<+++
    SfiI @50°C<++++--++++++
    SfoI+++++++++<+++++
    SgrAI<++<++++<+++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    SmaI @25°C<++++++<+++++++
    SmlI @55°C<+<++<+++
    SnaBI+++<+<+<+++++++
    SpeI (1) (2)++++++<++++++++
    SphI (1)+++++++++<+<+
    SphI-HF®+++++++<+++++++
    SrfI+<+++++++
    SspI (1)++++++++++++
    SspI-HF®+++++<+++++++
    StuI++++++<+<+++++++
    StyD4I<++<+++<+<+<+
    StyI (1)+<+<++<+<+
    StyI-HF®+++<+<++++++
    SwaI @25°C<+<+<+<+<++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    TaqαI @65°C+++++++
    TfiI @65°C<++<++<+<+++++
    TliI @75°C--<++++++++
    TseI @65°C++++++++++++++++++
    Tsp45I @65°C<++++--+<+
    Tsp509I @65°C++++++++++++++
    EnzymeTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    TspMI @75°C+++++++<+++++++
    TspRI @65°C++<+<+++++++
    Tth111I @65°C++++++++<+<++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    XbaI (2)++++++<+-++++
    XcmI+++++++<+++++++
    XhoI (2)<+<++++<++++++
    XmaI+++++++<+--
    XmnI++++++<+<++++++
    EnzymeBack to topTaq in std Taq
    Reaction Buffer
    Taq in ThermoPol™
    Reaction Buffer
    Phusion in
    Phusion HF Buffer
    Q5 in
    Q5 Buffer*
    OneTaq in
    OneTaq Buffer
    LongAmp Taq in
    LongAmp Taq Buffer
    ZraI++++++<+<++++