Thermostable RNase H requires Mg2+ for activity, therefore buffers containing MgCl2 may be suitable for Thermostable RNase H reactions. The optimal reaction buffer is 1X RNase H Reaction Buffer, but we have tested NEBuffers 1–4, NEBuffers r1.1–r3.1 and rCutSmart® Buffer:
| Buffer | 37°C | 50°C | 65°C | >65°C |
| RNase H Buffer | +++ | +++ | +++ | +++ |
| NEBuffer 1 | ++ | ++ | ++ | ++ |
| NEBuffer 2; | +++ | +++ | ++ | ++ |
| NEBuffer 3 | +++ | +++ | +++ | + |
| NEBuffer 4 | ++ | +++ | +++ | +++ |
| NEBuffer r1.1 | ++ | ++ | +++ | - |
| NEBuffer r2.1 | ++ | ++ | ++ | ++ |
| NEBuffer r3.1 | +++ | +++ | +++ | - |
| rCutSmart Buffer | ++ | ++ | ++ | + |
