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

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