Inactivation and residual activity of restriction enzymes
Restriction enzymes are commonly inactivated by a heat treatment after digestion is complete. However, heat tolerance varies between enzymes, and in some cases is insufficient to completely inactivate particular restriction enzymes. Therefore, we have tested four types of inactivation treatment to determine the best conditions for complete inactivation of each enzyme:
- Heating at 60°C for 15 minutes
- Heating at 70°C for 15 minutes
- Ethanol precipitation
- Phenol extraction
These tables list enzymes and their residual activity following each inactivation procedure.
| Restriction Enzyme |
60°C 15 min |
70°C 15 min |
Ethanol Precipitation |
Phenol Extraction |
|---|---|---|---|---|
| Aat II | + | – | – | NT |
| Acc I | + | – | – | NT |
| Acc II | + | + | – | NT |
| Acc III | NT | – | – | NT |
| Afa I | + | – | + | – |
| Afl II | – | – | – | – |
| Alu I | – | NT | + | – |
| Aor13H I | NT | + | + | – |
| Aor51H I | – | NT | – | NT |
| Apa I | – | NT | – | NT |
| ApaL I | + | – | – | NT |
| Ava II | + | – | + | – |
| Avi II | + | – | + | – |
| Bal I | – | – | – | – |
| BamH I | + | + | – | – |
| Ban II | – | NT | – | NT |
| Bcn I | + | + | – | NT |
| Bgl I | – | NT | + | – |
| Bgl II | + | – | + | – |
| Bln I | + | + | – | – |
| Bpu1102 I | + | + | + | – |
| Bsp1286 I | – | NT | – | NT |
| Bsp1407 I | + | – | + | – |
| BspT107 I | – | NT | – | NT |
| BssH II | + | – | – | NT |
| BstP I | NT | + | – | NT |
| BstX I | – | NT | – | NT |
| Bst1107 I | + | + | + | – |
| Cfr10 I | + | + | + | – |
| Cfr13 I | – | NT | – | NT |
| Cla I | + | – | – | – |
| Cpo I | – | NT | – | NT |
| Dra I | – | NT | – | NT |
| Dde I | + | – | – | – |
| Eae I | – | NT | – | NT |
| EcoO109 I | – | NT | – | NT |
| EcoO65 I | + | – | + | – |
| EcoR I | – | NT | – | NT |
| EcoR V | + | – | + | – |
| EcoT14 I | – | NT | – | NT |
| EcoT22 I | – | NT | – | NT |
| Eco52 I | – | NT | + | – |
| Eco81 I | + | – | – | NT |
| Fba I | + | + | – | NT |
| Fok I | – | NT | + | – |
| Fse I | – | NT | – | NT |
| Hae II | – | – | – | – |
| Hae III | + | – | – | NT |
| Hap II | + | – | + | – |
| Restriction Enzyme |
60°C 15 min |
70°C 15 min |
Ethanol Precipitation |
Phenol Extraction |
|---|---|---|---|---|
| Hha I | + | – | – | – |
| Hinc II | + | – | – | NT |
| Hind III | + | – | – | NT |
| Hinf I | + | – | – | NT |
| Hin1 I | – | NT | + | – |
| Hpa I | – | NT | – | NT |
| Kpn I | – | NT | – | NT |
| Mbo I | + | – | – | NT |
| Mbo II | + | – | – | NT |
| Mfl I | + | + | + | – |
| Mlu I | + | + | – | NT |
| Msp I | – | NT | + | – |
| Mun I | + | – | + | – |
| Nae I | – | NT | – | NT |
| Nco I | + | – | + | – |
| Nde I | – | – | – | – |
| Nhe I | + | – | – | NT |
| Not I | – | – | + | – |
| Nru I | – | NT | + | – |
| PmaC I | – | NT | – | NT |
| PshA I | – | NT | – | NT |
| PshB I | + | – | – | NT |
| Psp1406 I | – | NT | + | – |
| Pvu I | – | – | – | – |
| Pvu II | + | + | + | – |
| Sac I | – | NT | – | NT |
| Sac II | + | – | + | – |
| Sal I | – | – | – | – |
| Sau3A I | + | – | – | NT |
| Sca I | – | NT | – | NT |
| Sfi I | + | – | + | – |
| Sma I | – | NT | + | – |
| SnaB I | – | NT | – | NT |
| Spe I | – | NT | – | NT |
| Sph I | – | NT | – | NT |
| Sse8387 I | – | NT | – | NT |
| Ssp I | – | NT | – | NT |
| Stu I | – | NT | + | – |
| Taq I | NT | + | + | – |
| Tth 111 I | NT | + | – | NT |
| Van91 I | – | NT | + | – |
| VpaK11B I | – | NT | – | NT |
| Xba I | – | – | – | – |
| Xho I | + | + | – | – |
| Xsp I | + | + | + | – |
"+" indicates residual activity remaining, "–" indicates no residual activity, and "NT" indicates not tested
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