Protocol for cleavage with Thermostable RNase H (NEB #M0523)
Thermostable RNase H specifically recognizes and cleaves the phosphodiester bonds of an RNA strand in an RNA-DNA hybrid while leaving the DNA strand intact. The RNase H will only cleave the RNA in an RNA:DNA hybrid.
- Set-up a typical reaction as follows:
COMPONENTS 100 μl REACTION RNA:DNA duplex 2 µg RNase H Reaction Buffer(10X) 10 µl (1X) Thermostable RNase H 1 µl (5 units) Nuclease-free H2O (NEB#B1500S) up to 100 µl
- Incubate at 50°C for 20 minutes.
- The reaction can be stopped with addition of 1 µl of 0.5 M EDTA.
* Note: If targeting an RNA in a pool of RNA, we recommend titration of the enzyme to the intended substrate. Optimization may be required based on sequence, length and the type of reaction.