Heat Inactivation

Heat inactivation is a convenient method for stopping a restriction endonuclease reaction. Incubation at 65°C for 20 minutes inactivates the majority of restriction endonucleases that have an optimal incubation temperature of 37°C. Enzymes that cannot be inactivated at 65°C can often be inactivated by incubation at 80°C for 20 minutes. The table below indicates whether or not an enzyme can be heat inactivated and the temperature needed to do so.

For enzymes that cannot be heat-inactivated at 65°C or 80°C, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.

Heat inactivation was performed as follows to approximate a typical experiment. A 50 µl reaction mixture containing the appropriate NEBuffer, 0.5 µg of calf thymus DNA, and 5 or 10 µl of restriction endonuclease (at selling concentration) was incubated at 37°C for 60 minutes and then at 65°C or 80°C for 20 minutes. 0.5 µg of substrate DNA (usually lambda) was added to the reaction mixture and incubated at the optimal reaction temperature of the enzyme for 60 minutes. Any digestion (complete or partial) of the substrate DNA after the second incubation, as seen by agarose gel electrophoresis, was interpreted as incomplete heat inactivation.

Please also check these additional information about:
NEBuffer Performance | Activity at 37°C | Time-Saver Enzymes

Enzyme Heat Inactivation Temperature Heat Inactivation Time
AatII 80°C 20minutes
Acc65I 65°C 20minutes
AccI 80°C 20minutes
AciI 65°C 20minutes
AclI No --
AcuI 65°C 20minutes
AfeI 65°C 20minutes
AflII 65°C 20minutes
AflIII 80°C 20minutes
AgeI-HF® 65°C 20minutes
AhdI 65°C 20minutes
AleI 80°C 20minutes
AleI-v2 65°C 20minutes
AluI 80°C 20minutes
AlwI No --
AlwNI 80°C 20minutes
ApaI 65°C 20minutes
ApaLI No --
ApeKI No --
ApoI-HF® 80°C 20minutes
AscI 80°C 20minutes
AseI 65°C 20minutes
AsiSI 80°C 20minutes
AvaI 80°C 20minutes
AvaII 80°C 20minutes
AvrII No --
BaeGI 80°C 20minutes
BaeI 65°C 20minutes
BamHI No --
BamHI-HF® No --
BanI 65°C 20minutes
BanII 80°C 20minutes
BbsI 65°C 20minutes
BbsI-HF® 65°C 20minutes
BbvCI No --
BbvI 65°C 20minutes
BccI 65°C 20minutes
BceAI 65°C 20minutes
BcgI 65°C 20minutes
BciVI 80°C 20minutes
BclI No --
BclI-HF 65°C 20minutes
BcoDI No --
BfaI 80°C 20minutes
BfuAI 65°C 20minutes
BfuCI 80°C 20minutes
BglI 65°C 20minutes
BglII No --
BlpI No --
BmgBI 65°C 20minutes
BmrI 65°C 20minutes
BmtI-HF® 65°C 20minutes
BpmI 65°C 20minutes
Bpu10I 80°C 20minutes
BpuEI 65°C 20minutes
BsaAI No --
BsaBI 80°C 20minutes
BsaHI 80°C 20minutes
BsaI-HF® 65°C 20minutes
BsaI-HF®v2 80°C 20minutes
BsaJI 80°C 20minutes
BsaWI 80°C 20minutes
BsaXI No --
BseRI 80°C 20minutes
BseYI 80°C 20minutes
BsgI 65°C 20minutes
BsiEI No --
BsiHKAI No --
BsiWI 80°C 20minutes
BsiWI-HF® 80°C 20minutes
BslI No --
BsmAI No --
BsmBI-v2 80°C 20minutes
BsmFI 80°C 20minutes
BsmI 80°C 20minutes
BsoBI 80°C 20minutes
Bsp1286I 65°C 20minutes
BspCNI 80°C 20minutes
BspDI 80°C 20minutes
BspEI 80°C 20minutes
BspHI 80°C 20minutes
BspMI 65°C 20minutes
BspQI 80°C 20minutes
BsrBI 80°C 20minutes
BsrDI 80°C 20minutes
BsrFI No --
BsrFI-v2 No --
BsrGI-HF® 80°C 20minutes
BsrI 80°C 20minutes
BssHII 65°C 20minutes
BssKI 80°C 20minutes
BssSI 80°C 20minutes
BssSI-v2 No --
BstAPI 80°C 20minutes
BstBI No --
BstEII-HF® No --
BstNI No --
BstUI No --
BstXI 80°C 20minutes
BstYI No --
BstZ17I No --
BstZ17I-HF® No --
Bsu36I 80°C 20minutes
BtgI 80°C 20minutes
BtgZI 80°C 20minutes
BtsCI 80°C 20minutes
BtsI 80°C 20minutes
BtsI-v2 No --
BtsIMutI 80°C 20minutes
Cac8I 65°C 20minutes
ClaI 65°C 20minutes
CspCI 65°C 20minutes
CviKI-1 No --
CviQI No --
DdeI 65°C 20minutes
DpnI 80°C 20minutes
DpnII 65°C 20minutes
DraI 65°C 20minutes
DraIII 65°C 20minutes
DraIII-HF® No --
DrdI 65°C 20minutes
EaeI 65°C 20minutes
EagI-HF® 65°C 20minutes
EarI 65°C 20minutes
EciI 65°C 20minutes
Eco53kI 65°C 20minutes
EcoNI 65°C 20minutes
EcoO109I 65°C 20minutes
EcoP15I 65°C 20minutes
EcoRI 65°C 20minutes
EcoRI-HF® 65°C 20minutes
EcoRV 80°C 20minutes
EcoRV-HF® 65°C 20minutes
Esp3I 65°C 20minutes
FatI 80°C 20minutes
FauI 65°C 20minutes
Fnu4HI No --
FokI 65°C 20minutes
FseI 65°C 20minutes
FspI No --
HaeII 80°C 20minutes
HaeIII 80°C 20minutes
HgaI 65°C 20minutes
HhaI 65°C 20minutes
HinP1I 65°C 20minutes
HincII 65°C 20minutes
HindIII 80°C 20minutes
HindIII-HF® 80°C 20minutes
HinfI 80°C 20minutes
HpaI No --
HpaII 80°C 20minutes
HphI 65°C 20minutes
Hpy166II 65°C 20minutes
Hpy188I 65°C 20minutes
Hpy188III 65°C 20minutes
Hpy99I 65°C 20minutes
HpyAV 65°C 20minutes
HpyCH4III 65°C 20minutes
HpyCH4IV 65°C 20minutes
HpyCH4V 65°C 20minutes
I-CeuI 65°C 20minutes
I-SceI 65°C 20minutes
KasI 65°C 20minutes
KpnI-HF® No --
MboI 65°C 20minutes
MboII 65°C 20minutes
MfeI-HF® No --
MluCI No --
MluI-HF® No --
MlyI 65°C 20minutes
MmeI 65°C 20minutes
MnlI 65°C 20minutes
MscI 80°C 20minutes
MseI 65°C 20minutes
MslI 80°C 20minutes
MspA1I 65°C 20minutes
MspI No --
MspJI 65°C 20minutes
MwoI No --
NaeI No --
NarI 65°C 20minutes
Nb.BbvCI 80°C 20minutes
Nb.BsmI 80°C 20minutes
Nb.BsrDI 80°C 20minutes
Nb.BssSI No --
Nb.BtsI 80°C 20minutes
NciI No --
NcoI 80°C 20minutes
NcoI-HF® 80°C 20minutes
NdeI 65°C 20minutes
NgoMIV No --
NheI-HF® 80°C 20minutes
NlaIII 65°C 20minutes
NlaIV 65°C 20minutes
NmeAIII 65°C 20minutes
NotI 65°C 20minutes
NotI-HF® 65°C 20minutes
NruI-HF® No --
NsiI 65°C 20minutes
NsiI-HF® 80°C 20minutes
NspI 65°C 20minutes
Nt.AlwI 80°C 20minutes
Nt.BbvCI 80°C 20minutes
Nt.BsmAI 65°C 20minutes
Nt.BspQI 80°C 20minutes
Nt.BstNBI 80°C 20minutes
Nt.CviPII 65°C 20minutes
PI-PspI No --
PI-SceI 65°C 20minutes
PacI 65°C 20minutes
PaeR7I No --
PaqCI® 65°C 20minutes
PciI 80°C 20minutes
PflFI 65°C 20minutes
PflMI 65°C 20minutes
PhoI No --
PleI 65°C 20minutes
PluTI 65°C 20minutes
PmeI 65°C 20minutes
PmlI 65°C 20minutes
PpuMI No --
PshAI 65°C 20minutes
PsiI 65°C 20minutes
PsiI-v2 65°C 20minutes
PspGI No --
PspOMI 65°C 20minutes
PspXI No --
PstI 80°C 20minutes
PstI-HF® No --
PvuI-HF® No --
PvuII No --
PvuII-HF® No --
RsaI No --
RsrII 65°C 20minutes
SacI-HF® 65°C 20minutes
SacII 65°C 20minutes
SalI 65°C 20minutes
SalI-HF® 65°C 20minutes
SapI 65°C 20minutes
Sau3AI 65°C 20minutes
Sau96I 65°C 20minutes
SbfI-HF® 80°C 20minutes
ScaI 80°C 20minutes
ScaI-HF® 80°C 20minutes
ScrFI 65°C 20minutes
SexAI 65°C 20minutes
SfaNI 65°C 20minutes
SfcI 65°C 20minutes
SfiI No --
SfoI No --
SgrAI 65°C 20minutes
SmaI 65°C 20minutes
SmlI No --
SnaBI 80°C 20minutes
SpeI-HF® 80°C 20minutes
SphI 65°C 20minutes
SphI-HF® 65°C 20minutes
SrfI 65°C 20minutes
SspI-HF® 65°C 20minutes
StuI No --
StyD4I 65°C 20minutes
StyI-HF® 65°C 20minutes
SwaI 65°C 20minutes
TaqI-v2 No --
TfiI No --
TliI No --
TseI No --
Tsp45I No --
Tsp509I No --
TspMI No --
TspRI No --
Tth111I No --
WarmStart® Nt.BstNBI 80°C 20minutes
XbaI 65°C 20minutes
XcmI 65°C 20minutes
XhoI 65°C 20minutes
XmaI 65°C 20minutes
XmnI 65°C 20minutes
ZraI 80°C 20minutes

§ A HF version of this enzyme is available.
HF® is a registered trademark of New England Biolabs, Inc.