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  • Activity of Restriction Enzymes in a Q5®, Taq or Phusion PCR Mix

    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 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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp 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++++++++++++++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    AscI (2)++++++<+<+<+-
    AseI+++++<+++++
    AsiSI++++++++<+++++++
    AvaI+++++++++<+++++
    AvaII++++++++<+++++++
    AvrII++++++<+<+++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    BaeGI+++++++++<+++++++
    BaeI @25°C--++<+<+<+
    BamHI (1)+++++++++<+++++++
    BamHI-HF®++++++-<+<+++
    BanI+++++++++<+++++++
    BanII+++++++++<+++++++
    BbsI++++++<+<+++++++
    BbvCI<+++++--<+<+
    BbvI++++++++<+++++++
    BccI<+<+<+<+<+<+
    BceAI<+<+++<++<+
    BcgI+++<++
    BciVI----<+-
    BclI @50°C+++++++++++++++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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®++++<+<+<+
    BsaJI @60°C++++++++<+++++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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<+++++++<+++++++
    BspMI++++++<+<+<+
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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+++++++++<+++++++
    Bsu36I<+<+<+<+<++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    BtgI<+++++<+<+<+
    BtgZI @60°C+++++++++++++
    BtsαI @55°C-++++-++++++
    BtsCI @50°C++++++<+<+++++++
    BtsI @55°C++++++<+<+++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    Cac8I++++++<+<++++++
    ClaI+++++<+<+<+++
    CspCI<+<++-<+<+
    CviAII @25°C+++++++<+++++++
    CviKI-1-+++++<+++++++
    CviQI @25°C+++++++++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    DdeI+++++++++++++++
    DpnI<+++++++++++++
    DpnII++++++++++++++++
    DraI++++++++<+++++++
    DraIII (1)<+<+<+<+<+
    DraIII-HF®++++++++++++
    DrdI++++++++<+++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    EaeI++++++-<+<+<+
    EagI (1)<+<+++++++++++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    FatI @55°C+++++++<+<++++
    FauI @55°C++++++<++++++
    Fnu4HI++++++<+<++++
    FokI++++++++++++++
    FseI<++++<++++-
    FspI+<+++<+++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    HaeII+++++++++<+++++++
    HaeIII+++++++++<+++++++
    HgaI+++<++<+<++<++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    KasI-++++++<++++-
    KpnI (1) (2)+++++++++++<+
    KpnI-HF® (2)++++++-<+<+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp 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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    NaeI<+<++<+<+<+
    NarI--++<+++++++
    NciI++++++<+<++<+
    NcoI (1) (2)+++++++<+++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    PacI (2)++++++<+<++++++
    PaeR7I++++++<+<+++++++
    PciI+<+-<+--
    PflFI++++++<+<+<++
    PflMI+++++<++++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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 BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    RsaI++++++++<+++++++
    RsrII<++<++--<+<+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    SacI (1)+++++++<+++++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    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<++<++++<+++++
    SmaI @25°C<++++++<+++++++
    SmlI @55°C<+<++<+++
    EnzymeTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    SnaBI+++<+<+<+++++++
    SpeI (1) (2)++++++<++++++++
    SphI (1)+++++++++<+<+
    SphI-HF®+++++++<+++++++
    SspI (1)++++++++++++
    SspI-HF®+++++<+++++++
    StuI++++++<+<+++++++
    StyD4I<++<+++<+<+<+
    StyI (1)+<+<++<+<+
    StyI-HF®+++<+<++++++
    SwaI @25°C<+<+<+<+<++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    TaqαI @65°C+++++++
    TfiI @65°C<++<++<+<+++++
    TliI @75°C--<++++++++
    TseI @65°C++++++++++++++++++
    Tsp45I @65°C<++++--+<+
    Tsp509I @65°C++++++++++++++
    TspMI @75°C+++++++<+++++++
    TspRI @65°C++<+<+++++++
    Tth111I @65°C++++++++<+<++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    XbaI (2)++++++<+-++++
    XcmI+++++++<+++++++
    XhoI (2)<+<++++<++++++
    XmaI+++++++<+--
    XmnI++++++<+<++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferQ5 in Q5 Buffer*OneTaq in OneTaq BufferLongAmp Taq in LongAmp Taq Buffer
    ZraI++++++<+<++++

    (1) A HF version of this enzyme is available.
    (2) A RE-Mix version of this enzyme is available.

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