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.

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 Activity | Activity at 37°C | Time-Saver Enzymes

EnzymeHeat Inactivation TemperatureHeat Inactivation Time
AatII80°C20 min
AbaSI65°C20 min
Acc65I65°C20 min
AccI80°C20 min
AciI65°C20 min
AclINo--
AcuI65°C20 min
AfeI65°C20 min
AflII65°C20 min
AflIII80°C20 min
AgeI§65°C20 min
AgeI-HF®65°C20 min
AhdI65°C20 min
AleI80°C20 min
AluI80°C20 min
AlwINo--
AlwNI80°C20 min
ApaI65°C20 min
ApaLINo--
ApeKINo--
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
ApoI§80°C20 min
ApoI-HF80°C20 min
AscI80°C20 min
AseI65°C20 min
AsiSI80°C20 min
AvaI80°C20 min
AvaII80°C20 min
AvrIINo--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
BaeGI80°C20 min
BaeI65°C20 min
BamHI§No--
BamHI-HF®No--
BanI65°C20 min
BanII80°C20 min
BbsI65°C20 min
BbsI-HF®65°C20 min
BbvCINo--
BbvI65°C20 min
BccI65°C20 min
BceAI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
BcgI65°C20 min
BciVI80°C20 min
BclINo--
BcoDINo--
BfaI80°C20 min
BfuAI65°C20 min
BfuCI80°C20 min
BglI65°C20 min
BglIINo--
BlpINo--
BmgBI65°C20 min
BmrI65°C20 min
BmtI§65°C20 min
BmtI-HF®65°C20 min
BpmI65°C20 min
Bpu10I80°C20 min
BpuEI65°C20 min
BsaAINo--
BsaBI80°C20 min
BsaHI80°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
BsaI§65°C20 min
BsaI-HF®65°C20 min
BsaJI80°C20 min
BsaWI80°C20 min
BsaXINo--
BseRI80°C20 min
BseYI80°C20 min
BsgI65°C20 min
BsiEINo--
BsiHKAINo--
BsiWI65°C20 min
BsiWI-HF®No--
BslINo--
BsmAINo--
BsmBI80°C20 min
BsmFI80°C20 min
BsmI80°C20 min
BsoBI80°C20 min
Bsp1286I65°C20 min
BspCNI80°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
BspDI80°C20 min
BspEI80°C20 min
BspHI80°C20 min
BspMI65°C20 min
BspQI80°C20 min
BsrBI80°C20 min
BsrDI80°C20 min
BsrFαINo--
BsrFINo--
BsrGI§80°C20 min
BsrGI-HF®80°C20 min
BsrI80°C20 min
BssHII65°C20 min
BssKI80°C20 min
BssSαINo--
BssSI80°C20 min
BstAPI80°C20 min
BstBINo--
BstEII §No--
BstEII-HF®No--
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
BstNINo--
BstUINo--
BstXI80°C20 min
BstYINo--
BstZ17INo--
BstZ17I-HF®No--
Bsu36I80°C20 min
BtgI80°C20 min
BtgZI80°C20 min
BtsαINo--
BtsCI80°C20 min
BtsI80°C20 min
BtsIMutI80°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
Cac8I65°C20 min
ClaI65°C20 min
CspCI65°C20 min
CviAII65°C20 min
CviKI-1No--
CviQINo--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
DdeI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
DpnI80°C20 min
DpnII65°C20 min
DraI65°C20 min
DraIII§65°C20 min
DraIII-HF®No--
DrdI65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
EaeI65°C20 min
EagI§65°C20 min
EagI-HF®65°C20 min
EarI65°C20 min
EciI65°C20 min
Eco53kI65°C20 min
EcoNI65°C20 min
EcoO109I65°C20 min
EcoP15I65°C20 min
EcoRI§65°C20 min
EcoRI-HF®65°C20 min
EcoRV§80°C20 min
EcoRV-HF®65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
FatI80°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
FauI65°C20 min
Fnu4HINo--
FokI65°C20 min
FseI65°C20 min
FspEI80°C20 min
FspINo--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
HaeII80°C20 min
HaeIII80°C20 min
HgaI65°C20 min
HhaI65°C20 min
HincII65°C20 min
HindIII§80°C20 min
HindIII-HF®80°C20 min
HinfI80°C20 min
HinP1I65°C20 min
HpaINo--
HpaII80°C20 min
HphI65°C20 min
Hpy166II65°C20 min
Hpy188I65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
Hpy188III65°C20 min
Hpy99I65°C20 min
HpyAV65°C20 min
HpyCH4III65°C20 min
HpyCH4IV65°C20 min
HpyCH4V65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
I-CeuI65°C20 min
I-SceI65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
KasI65°C20 min
KpnI§No--
KpnI-HF®No--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
LpnPI65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
MboI65°C20 min
MboII65°C20 min
MfeI§No--
MfeI-HF®No--
MluCINo--
MluI§80°C20 min
MluI-HF®No--
MlyI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
MmeI65°C20 min
MnlI65°C20 min
MscI80°C20 min
MseI65°C20 min
MslI80°C20 min
MspA1I65°C20 min
MspINo--
MspJI65°C20 min
MwoINo--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
NaeINo--
NarI65°C20 min
Nb.BbvCI80°C20 min
Nb.BsmI80°C20 min
Nb.BsrDI80°C20 min
Nb.BssSINo--
Nb.BtsI80°C20 min
NciINo--
NcoI§80°C20 min
NcoI-HF®80°C20 min
NdeI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
NgoMIVNo--
NheI§65°C20 min
NheI-HF®80°C20 min
NlaIII65°C20 min
NlaIV65°C20 min
NmeAIII65°C20 min
NotI§65°C20 min
NotI-HF®65°C20 min
NruI§No--
NruI-HF®No--
NsiI§65°C20 min
NsiI-HF®80°C20 min
NspI65°C20 min
Nt.AlwI80°C20 min
Nt.BbvCI80°C20 min
Nt.BsmAI65°C20 min
Nt.BspQI80°C20 min
Nt.BstNBI80°C20 min
Nt.CviPII65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
PacI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
PaeR7INo--
PciI80°C20 min
PflFI65°C20 min
PflMI65°C20 min
PhoINo--
PI-PspINo--
PI-SceI65°C20 min
PleI65°C20 min
PluTI65°C20 min
PmeI65°C20 min
PmlI65°C20 min
PpuMINo--
PshAI65°C20 min
PsiI65°C20 min
PspGINo--
PspOMI65°C20 min
PspXINo--
PstI§80°C20 min
PstI-HF®No--
PvuI§No--
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
PvuI-HF®No--
PvuII§No--
PvuII-HF®No--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
RsaINo--
RsrII65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
SacI§65°C20 min
SacI-HF®65°C20 min
SacII65°C20 min
SalI§65°C20 min
SalI-HF®65°C20 min
SapI65°C20 min
Sau3AI65°C20 min
Sau96I65°C20 min
SbfI§80°C20 min
SbfI-HF®80°C20 min
ScaI§80°C20 min
ScaI-HF®80°C20 min
ScrFI65°C20 min
SexAI65°C20 min
SfaNI65°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
SfcI65°C20 min
SfiINo--
SfoINo--
SgrAI65°C20 min
SmaI65°C20 min
SmlINo--
SnaBI80°C20 min
SpeI§80°C20 min
SpeI-HF®80°C20 min
SphI§65°C20 min
SphI-HF®65°C20 min
SrfI65°C20 min
SspI§65°C20 min
SspI-HF®65°C20 min
StuINo--
StyD4I65°C20 min
StyI§65°C20 min
StyI-HF®65°C20 min
SwaI65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
TaqαI80°C20 min
EnzymeHeat Inactivation TemperatureHeat Inactivation Time
TfiINo--
TliINo--
TseINo--
Tsp45INo--
Tsp509INo--
TspMINo--
TspRINo--
Tth111INo--
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
XbaI65°C20 min
XcmI65°C20 min
XhoI65°C20 min
XmaI65°C20 min
XmnI65°C20 min
EnzymeBack to topHeat Inactivation TemperatureHeat Inactivation Time
ZraI80°C20 min
(1) A HF version of this enzyme is available.
(2) A RE-Mix version of this enzyme is available.
HF® is a registered trademark of New England Biolabs, Inc.