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  • Activity of Restriction Enzymes in a 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 or Phusion® 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.

    Please also check these additional information about:
    PCR MixRActivity of Restriction Enzymes in a Vent

    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 BufferTaq in Crimson Taq Buffer
    AatII<++<++++++
    Acc65I++++++<+<+
    AccI++<++<+++
    AciI+++++++
    AclI++++++<++++
    AcuI+++++++++++
    AfeI+++++++++++
    AflII<+<+<+<+
    AflIII<+<+++
    AgeI+++++++++++
    AhdI+++<+-++
    AleI++++++<+++
    AluI++++++++++++
    AlwI<++-<+<+
    AlwNI<++<+++++<+
    ApaI @25°C++++++<+++
    ApaLI++++++<++++
    ApeKI @75°C<++<+++++++
    ApoI @50°C++++++++++++
    AscI++++++<++++
    AseI++++++
    AsiSI++++++++++
    AvaI++++++++++++
    AvaII+++++++++++
    AvrII++++++<++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    BaeGI++++++++++++
    BaeI @25°C--++++
    BamHI++++++++++
    BamHI-HF®++++++-+
    BanI++++++++++++
    BanII++++++++++++
    BbsI++++++<++++
    BbvCI<+++++-+++
    BbvI+++++++++++
    BccI<+<+<+<+
    BceAI<+<+++++
    BcgI+++<+++
    BciVI---+
    BclI @50°C++++++++++++
    BfaI<++--<+
    BfuAI @50°C<++<++++
    BfuCI<+<+<+<+
    BglI<++<++<+
    BglII<+<++++
    BlpI<++<++<+<+
    BmgBI<+-++
    BmrI<++<+++++++
    BmtI++++++++++
    BpmI<+<+++++++
    Bpu10I<+<+++++
    BpuEI++++++++++
    BsaAI+++++++++++
    BsaBI @60°C<+<++++++
    BsaHI++++++++++++
    BsaI+++<++++
    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<++++++++++
    BspMI++++++++
    BspQI @50°C<++++++++
    BsrBI++++++++++
    BsrDI @65°C<+<++++
    BsrFI<+<+++++++
    BsrGI<+<++++++
    BsrI @65°C++++++++++
    BssHII @50°C+++++++++
    BssKI @60°C++++++<+<+
    BssSI++++++++++++
    BstBI @65°C++++++++++++
    BstEII  @60°C++++++<+<+
    BstNI @60°C++++++<++
    BstUI @60°C++++++<++++
    BstXI<++<++++
    BstYI @60°C++++++<++++
    BstZ17I++++++++++++
    Bsu36I<+<+<+<+
    BtgI<++++++
    BtgZI @60°C++++++++++
    BtsCI @50°C++++++<++++
    BtsI @55°C++++++<++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson 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 BufferTaq in Crimson Taq Buffer
    DdeI+++++++++
    DpnI<+++++++++
    DpnII++++++++++++
    DraI+++++++++++
    DrdI+++++++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    EaeI++++++-+++
    EagI<+<+++++++
    EagI-HF®+++++++
    EarI++++++++++
    EciI<+<++++++
    EcoNI<+++++++++
    EcoO109I++++++-+++
    EcoP15I<+<++-
    EcoRI++++++
    EcoRI-HF®++++++++++
    EcoRV<+<++<+
    EcoRV-HF®++<+<+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson 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 BufferTaq in Crimson Taq Buffer
    HaeII++++++++++++
    HaeIII++++++++++++
    HgaI+++<+++++
    HhaI++++++++++++
    HincII++++++<++++
    HindIII++++++++
    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 BufferTaq in Crimson Taq Buffer
    KasI-+++++++++
    KpnI++++++++++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    MboI++++++++++++
    MboII+++++++++++
    MfeI+++++<++
    MfeI-HF®++-+
    MluI+++++++++
    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 BufferTaq in Crimson Taq Buffer
    NaeI<+<+++
    NarI--++++
    NciI++++++<++
    NcoI+++++++++
    NcoI-HF®++++++-<+
    NdeI++<+++++++
    NgoMIV--++
    NheI++++++<++
    NheI-HF®++++++-+++
    NlaIII+++<++++
    NlaIV<++++++++
    NmeAIII<+<+++++++
    NotI+++++++
    NotI-HF®+++++<+++
    NruI++++++
    NsiI++++++++++++
    NspI++++++<++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    PacI++++++<+<+
    PaeR7I++++++<++++
    PciI+<+-+++
    PflFI++++++<++++
    PflMI++++++++
    PhoI @75°C<++++++++++
    PleI+<+<+++
    PmeI++++++<+++
    PmlI+--+++
    PpuMI++++++++++++
    PshAI++++++<++
    PsiI+++++<+++
    PspGI @75°C++++++++++++
    PspOMI++++++++++
    PspXI+++++++++++
    PstI+++++<+
    PvuI<+<++++<+
    PvuII++++++++++
    PvuII-HF®+++-+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    RsaI+++++++++++
    RsrII<++<++-+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    SacI++++++++
    SacI-HF®++++++<++++
    SacII++++++++++++
    SalI<+<+++<+
    SalI-HF®++++++++++
    SapI<++<++++++
    Sau3AI++++++<++
    Sau96I<++<++++
    SbfI<++<++++
    SbfI-HF®++-<+
    ScaI<++<++++++++
    ScaI-HF®++<++++
    ScrFI++++++++++++
    SexAI++++++++++++
    SfaNI--++++
    SfcI++++++<+<+
    SfiI @50°C<++++-<+
    SfoI++++++++++++
    SgrAI<++<++++++
    SmaI @25°C<+++++++++
    SmlI @55°C<+<++<+
    SnaBI+++<+<++
    SpeI++++++<++
    SphI-HF®+++++++++
    SphI++++++++++
    SspI++++++++++
    SspI-HF®+++++++
    StuI++++++<++++
    StyD4I<++<++++
    StyI+<+<++
    SwaI @25°C<+<+<+<+
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    TaqαI @65°C++++++++++
    TfiI @65°C<++<++<++++
    TseI @65°C++++++++++++
    Tsp45I @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 BufferTaq in Crimson Taq Buffer
    XbaI++++++<+++
    XcmI++++++++++
    XhoI<+<+++++++
    XmaI++++++++
    XmnI++++++<+++
    EnzymeBack to topTaq in std Taq Reaction BufferTaq in ThermoPol™ Reaction BufferPhusion in Phusion HF BufferTaq in Crimson Taq Buffer
    ZraI++++++<+++

    Phusion® was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific.
    Phusion® is a registered trademark of Thermo Fisher Scientific.
    ThermoPol™ is a trademark of New England Biolabs, Inc.