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热失活是终止酶切反应的一种简便易行的方法。大多数最适反应温度是37℃的酶在65℃下温育20分钟即可失活。一些在65℃下不能失活的酶如将温度升高至80℃，温育20分钟也可失活。下表注明了每种酶的失活条件。
在50μl的反应体系中，37℃条件下，以0.5μg小牛胸腺DNA作为底物，加入5-10μl内切酶及相应缓冲液，反应60分钟，然后升温至65℃或80℃温育20分钟进行热失活。在上述混合液中加入0.5μg底物DNA（通常为λDNA），调节温度至最适反应温度，温育60分钟。若琼脂糖电泳显示底物部分或完全被消化，则表明该酶没有完全被热失活。
| Enzyme |
Heat Inactivation (temperature) |
| Aat II |
Yes (65°C) |
| Acc I |
Yes (80°C) |
| Acc65 I |
Yes (65°C) |
| Aci I |
Yes (65°C) |
| Acl I |
No |
| Acu I |
Yes (65°C) |
| Afe I |
Yes (65°C) |
| Afl II |
Yes (65°C) |
| Afl III |
Yes (80°C) |
| Age I |
Yes (65°C) |
| Ahd I |
Yes (65°C) |
| Ale I |
Yes (65°C) |
| Alu I |
Yes (65°C) |
| Alw I |
Yes (65°C) |
| AlwN I |
Yes (65°C) |
| Apa I |
Yes (65°C) |
| ApaL I |
No |
| ApeK I |
No |
| Apo I |
Yes (80°C) |
| Asc I |
Yes (65°C) |
| Ase I |
Yes (65°C) |
| AsiS I |
Yes (80°C) |
| Ava I |
Yes (80°C) |
| Ava II |
Yes (65°C) |
| Avr II |
No |
| Bae I |
Yes (65°C) |
| BamH I |
No |
| Ban I |
Yes (65°C) |
| Ban II |
Yes (65°C) |
| Bbs I |
Yes (65°C) |
| Bbv I |
Yes (65°C) |
| BbvC I |
Yes (80°C) |
| Bcc I |
Yes (65°C) |
| BceA I |
Yes (65°C) |
| Bcg I |
Yes (65°C) |
| BciV I |
Yes (65°C) |
| Bcl I |
No |
| Bfa I |
Yes (80°C) |
| BfrB I |
Yes (80°C) |
| BfuA I |
Yes (65°C) |
| BfuC I |
Yes (80°C) |
| Bgl I |
Yes (65°C) |
| Bgl II |
No |
| Blp I |
No |
| Bme1580 I |
Yes (80°C) |
| BmgB I |
Yes (65°C) |
| Bmr I |
Yes (65°C) |
| Bmt I |
Yes (65°C) |
| Bpm I |
Yes (65°C) |
| Bpu10 I |
Yes (80°C) |
| BpuE I |
Yes (65°C) |
| Bsa I |
Yes (65°C) |
| BsaA I |
Yes (80°C) |
| BsaB I |
Yes (80°C) |
| BsaH I |
Yes (80°C) |
| BsaJ I |
Yes (80°C) |
| BsaW I |
Yes (80°C) |
| BsaX I |
No |
| BseR I |
Yes (65°C) |
| BseY I |
Yes (65°C) |
| Bsg I |
Yes (65°C) |
| BsiE I |
Yes (80°C) |
| BsiHKA I |
Yes (80°C) |
| BsiW I |
Yes (80°C) |
| Bsl I |
Yes (80°C) |
| Bsm I |
Yes (80°C) |
| BsmA I |
Yes (80°C) |
| BsmB I |
Yes (80°C) |
| BsmF I |
Yes (80°C) |
| BsoB I |
No |
| Bsp1286 I |
Yes (65°C) |
| BspCN I |
Yes (80°C) |
| BspD I |
Yes (65°C) |
| BspE I |
Yes (80°C) |
| BspH I |
Yes (65°C) |
| BspM I |
Yes (65°C) |
| Bsr I |
Yes (80°C) |
| BsrB I |
Yes (80°C) |
| BsrD I |
Yes (80°C) |
| BsrF I |
No |
| BsrG I |
Yes (80°C) |
| BssH II |
Yes (80°C) |
| BssK I |
Yes (80°C) |
| BssS I |
Yes (80°C) |
| BstAP I |
Yes (80°C) |
| BstB I |
No |
| BstE II |
No |
| BstF5 I |
No |
| BstN I |
No |
| BstU I |
No |
| BstX I |
Yes (65°C) |
| BstY I |
Yes (80°C) |
| BstZ17 I |
No |
| Bsu36 I |
Yes (80°C) |
| Btg I |
Yes (80°C) |
| BtgZ I |
Yes (80°C) |
| Bts I |
Yes (80°C) |
| Cac8 I |
Yes (65°C) |
| Cla I |
Yes (65°C) |
| CviA II |
Yes (65°C) |
| Dde I |
Yes (65°C) |
| Dpn I |
Yes (80°C) |
| Dpn II |
Yes (65°C) |
| Dra I |
Yes (65°C) |
| Dra III |
Yes (65°C) |
| Drd I |
Yes (65°C) |
| Eae I |
Yes (65°C) |
| Eag I |
Yes (65°C) |
| Ear I |
Yes (65°C) |
| Eci I |
Yes (65°C) |
| EcoN I |
Yes (65°C) |
| EcoO109 I |
Yes (65°C) |
| EcoR I |
Yes (65°C) |
| EcoR V |
Yes (80°C) |
| Fat I |
Yes (65°C) |
| Fau I |
Yes (65°C) |
| Fnu4H I |
Yes (65°C) |
| Fok I |
Yes (65°C) |
| Fse I |
Yes (65°C) |
| Fsp I |
Yes (65°C) |
| Hae II |
Yes (80°C) |
| Hae III |
Yes (80°C) |
| Hga I |
Yes (65°C) |
| Hha I |
Yes (65°C) |
| HinP1 I |
Yes (65°C) |
| Hinc II |
Yes (65°C) |
| Hind III |
Yes (65°C) |
| Hinf I |
Yes (80°C) |
| Hpa I |
No |
| Hpa II |
Yes (65°C) |
| Hph I |
Yes (65°C) |
| Hpy99 I |
Yes (65°C) |
| Hpy188 I |
Yes (65°C) |
| Hpy188 III |
Yes (65°C) |
| HpyCH4 III |
Yes (80°C) |
| HpyCH4 IV |
Yes (65°C) |
| HpyCH4 V |
Yes (65°C) |
| I-Ceu I |
Yes (65°C) |
| I-Sce I |
Yes (65°C) |
| Kas I |
Yes (65°C) |
| Kpn I |
No |
| Mbo I |
Yes (65°C) |
| Mbo II |
Yes (65°C) |
| McrBC |
Yes (65°C) |
| Mfe I |
Yes (65°C) |
| Mlu I |
Yes (65°C) |
| Mly I |
Yes (65°C) |
| Mme I |
Yes (80°C) |
| Mnl I |
Yes (65°C) |
| Msc I |
Yes (65°C) |
| Mse I |
Yes (65°C) |
| Msl I |
Yes (65°C) |
| Msp I |
Yes (65°C) |
| MspA1 I |
Yes (65°C) |
| Mwo I |
No |
| Nae I |
Yes (65°C) |
| N.Alw I |
Yes (80°C) |
| Nar I |
Yes (65°C) |
| N.BbvC IA |
Yes (80°C) |
| N.BbvC IB |
Yes (80°C) |
| N.BstNB I |
Yes (80°C) |
| Nci I |
No |
| Nco I |
Yes (65°C) |
| Nde I |
Yes (65°C) |
| NgoM IV |
Yes (80°C) |
| Nhe I |
Yes (65°C) |
| Nla III |
Yes (65°C) |
| Nla IV |
Yes (65°C) |
| Not I |
Yes (65°C) |
| Nru I |
Yes (65°C) |
| Nsi I |
Yes (65°C) |
| Nsp I |
Yes (65°C) |
| Pac I |
Yes (65°C) |
| PaeR7 I |
No |
| Pci I |
Yes (65°C) |
| PflF I |
Yes (65°C) |
| PflM I |
Yes (65°C) |
| Pho I |
No |
| PI-Psp I |
No |
| Pl-Sce I |
Yes (65°C) |
| Ple I |
Yes (65°C) |
| Pme I |
Yes (65°C) |
| Pml I |
Yes (65°C) |
| PpuM I |
No |
| PshA I |
Yes (65°C) |
| Psi I |
Yes (65°C) |
| PspG I |
No |
| PspOM I |
Yes (65°C) |
| Pst I |
Yes (80°C) |
| Pvu I |
Yes (80°C) |
| Pvu II |
No |
| Rsa I |
Yes (65°C) |
| Rsr II |
Yes (65°C) |
| Sac I |
Yes (65°C) |
| Sac II |
Yes (65°C) |
| Sal I |
Yes (65°C) |
| Sap I |
Yes (65°C) |
| Sau96 I |
Yes (80°C) |
| Sau3A I |
Yes (65°C) |
| Sbf I |
Yes (65°C) |
| Sca I |
Yes (80°C) |
| ScrF I |
Yes (65°C) |
| SexA I |
Yes (65°C) |
| SfaN I |
Yes (65°C) |
| Sfc I |
Yes (65°C) |
| Sfi I |
No |
| Sfo I |
Yes (65°C) |
| SgrA I |
Yes (65°C) |
| Sma I |
Yes (65°C) |
| Sml I |
No |
| SnaB I |
Yes (80°C) |
| Spe I |
Yes (65°C) |
| Sph I |
Yes (65°C) |
| Ssp I |
Yes (65°C) |
| Stu I |
Yes (65°C) |
| Sty I |
Yes (65°C) |
| StyD4 I |
Yes (65°C) |
| Swa I |
Yes (65°C) |
| Taq I |
Yes (80°C) |
| Tfi I |
No |
| Tli I |
No |
| Tse I |
No |
| Tsp45 I |
No |
| Tsp509 I |
No |
| TspR I |
No |
| Tth111 I |
No |
| Xba I |
Yes (65°C) |
| Xcm I |
Yes (65°C) |
| Xho I |
Yes (65°C) |
| Xma I |
Yes (65°C) |
| Xmn I |
Yes (65°C) |
| Zra I |
Yes (80°C) |
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