内切酶的热失活参数
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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) |