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

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

§ An 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.