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