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

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