New England Biolabs: Reagents for the Life Sciences Industry

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 §+++++++++++++<+
AgeI-HF®++++++++<+++++++
AhdI+++<+--<+<+
AleI++++++<+<+++<+
AluI++++++++++++++++
AlwI<++-<+<+<+<+
AlwNI<++<++++++<++
ApaI @25°C++++++<+<+<+-
ApaLI++++++<+<+++++++
ApeKI @75°C<++<+++++++<++
ApoI @50°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
ApoI-HF+++++++++++++++
AscI++++++<+<+<+-
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 §+++++++++<+++++++
BamHI-HF®++++++-<+<+++
BanI+++++++++<+++++++
BanII+++++++++<+++++++
BbsI++++++<+<+++++++
BbsI-HF®-+---+
BbvCI<+++++--<+<+
BbvI++++++++<+++++++
BccI<+<+<+<+<+<+
BceAI<+<+++<++<+
BcgI+++<++
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
BciVI----<+-
BclI @50°C+++++++++++++++++
BcoDI<+<++<++<+
BfaI<++--<+--
BfuAI @50°C<++<+++-<+-
BfuCI<+<+<+<+<+
BglI<++<++++<+<+
BglII<+<++++<+<+
BlpI<++<++<+<+<+-
BmgBI<+-+++<+<+
BmrI<++<+++++<+++++++
BmtI §++++++++<+++++++
BmtI-HF®+++++<++++++
BpmI<+<++++<+<++<++
Bpu10I<+<++++<++++++
BpuEI++++++++-<++<++
BsaAI++++++++++++++++
BsaBI @60°C<++++<++++++
BsaHI++++++++++++++++
BsaI §+++<+++-+
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
BsaI-HF®++++<+<+<+
BsaJI @60°C++++++++<+++++++
BsaWI @60°C<++<++++<+++
BsaXI<++<++<+<+<+<+
BseRI++++++++<++++
BseYI++++++++++++++++
BsgI<+<++<+<+<+
BsiEI @60°C++++++++<+++++
BsiHKAI @65°C<+-+++--
BsiWI @55°C++++++++<+++++++
BsiWI-HF®------
BslI @55°C+++++++++++++++++
BsmAI @55°C+++++++++++<+<+
BsmBI @55°C<++<+++++<+<+
BsmFI @65°C<+<++++++++
BsmI @65°C++++++<++++++
BsoBI+++++++++++++++++
Bsp1286I+<+<+<+<+<+
BspCNI @25°C<+<++<+--
BspDI<++<++++<+++++++
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
BspEI--<+<+--
BspHI<+++++++<+++++++
BspMI++++++<+<+<+
BspQI @50°C<+++++++++++++
BsrBI+++++++<+++++++
BsrDI @65°C<+<++
BsrFI<+<++++<++++
BsrGI §<+<+++++<++++
BsrI @65°C+++++++++<++++++
BssHII @50°C++++++<+++++++
BssKI @60°C++++++<+<+++++
BssSαI+++++++++<+++++++
BssSI+++++++++<++++++
BstAPI @60°C<+
BstBI @65°C+++++++++++++++++
BstEII  @60°C §++++++<+<+++++++
BstEII-HF®++++<+<+++++
BstNI @60°C++++++<+<+<+<+
BstUI @60°C++++++<+<+++++
BstXI<++<+++++<+
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
BstYI @60°C++++++<+<++++
BstZ17I+++++++++<+++++++
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 §<+<+<+<+<+
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
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 §<+<+++++++++++++
EagI-HF®+++++<+++++
EarI+++++++<++<+
EciI<+<++++++<++<++
Eco53kI<+
EcoNI<++++++<+++++++
EcoO109I++++++-<+<++
EcoP15I<+<++<+<++
EcoRI §+++++<+--
EcoRI-HF®+++++++<+++++++
EcoRV §<+<++<+-<+
EcoRV-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
FatI @55°C+++++++<+<++++
FauI @55°C++++++<++++++
Fnu4HI++++++<+<++++
FokI++++++++++++++
FseI<++++<++++-
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
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+++++++++<+++++++
HaeIII+++++++++<+++++++
HgaI+++<++<+<++<++
HhaI+++++++++<+++++++
HincII++++++<+<+++++++
HindIII §++++++<++++++
HindIII-HF®++++<+<+++++++
HinfI++++++++++++++++
HinP1I++++++++++++++++
HpaI+++++++++<+++++++
HpaII++++<+<+<+<+
HphI+++<++<+<+<+<+
Hpy166II+++++++++++++
Hpy188I+++++++<+++++
Hpy188III<++<+<++<+
Hpy99I+++++++-<+<+
HpyAV++++++-+++
HpyCH4III<++<+++<+<++<++
HpyCH4IV+++++<+<+++++++
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
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-++++++<++++-
KpnI §+++++++++++<+
KpnI-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
MboI+++++++++<+++++++
MboII+++++++++++
MfeI §+++++<+<+++++
MfeI-HF®++--+++<+
MluCI++++<+<++++
MluI §+++++++++++++
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--++<+++++++
NciI++++++<+<++<+
NcoI §+++++++<+++++
NcoI-HF®++++++-<++++
NdeI++<+++++++++<+
NgoMIV--+<+<+<+
NheI §++++++<+<+++++++
NheI-HF®++++++-<+++++
NlaIII+++<++<+++<+
NlaIV<+++++++<+++++++
NmeAIII<+<++++-<+<+
NotI §++++++<+<+<+
NotI-HF®+++++<+<+<++
NruI §++++++++++
NruI-HF®+++++--+-
NsiI §+++++++++++++
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++++++<+<++++++
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
PaeR7I++++++<+<+++++++
PciI+<+-<+--
PflFI++++++<+<+<++
PflMI+++++<++++++
PhoI @75°C<++++++++++++++++
PleI++++<+<+<+<+
PluTI<+
PmeI++++++<+<+++++++
PmlI+---+<+
PpuMI+++++++++<+++++++
PshAI++++++<+<+<+<+
PsiI+++++<+<+<+
PspGI @75°C++++++++++++++++++
PspOMI+++++++<+++++++
PspXI++++++++<+++++++
PstI §++++++<+<+
PstI-HF®+++++++<++++
PvuI §<+<++++<+-<+
PvuI-HF®+++++++++<++++++
PvuII §+++++++<+++++++
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
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++++++++<+++++++
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 §+++++++<+++++
SacI-HF®++++++<+<+<+++
SacII+++++++++<++++
SalI §<+<++++--
SalI-HF®+++++++<+++++
SapI<++<++++<+++++
Sau3AI++++++<+<+<+<+
Sau96I<++<++++++++++
SbfI §<++<+++<+<++++
SbfI-HF®++--<+<+
ScaI §<++<++++++++<+<+
ScaI-HF®++<+<+--
ScrFI++++++++++++++++++
SexAI+++++++++<+++++++
SfaNI--++<+<++<++
SfcI++++++<+<+++
SfiI @50°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
SfoI+++++++++<+++++
SgrAI<++<++++<+++++
SmaI @25°C<++++++<+++++++
SmlI @55°C<+<++<+++
SnaBI+++<+<+<+++++++
SpeI §++++++<++++++++
SphI §+++++++++<+<+
SphI-HF®+++++++<+++++++
SrfI+<+++++++
SspI §++++++++++++
SspI-HF®+++++<+++++++
StuI++++++<+<+++++++
StyD4I<++<+++<+<+<+
StyI §+<+<++<+<+
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++++++++++++++++++
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
Tsp45I @65°C<++++--+<+
Tsp509I @65°C++++++++++++++
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++++++<+-++++
XcmI+++++++<+++++++
XhoI<+<++++<++++++
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++++++<+<++++

§ A HF version of this enzyme is available.

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.