Protocol for cleavage with RNase H (NEB #M0297)

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.

  1. Set-up a typical reaction as follows:
    RNA:DNA duplex 2 µg
    RNase H Reaction Buffer(10X) 10 µl (1X)
    RNase H 1 µl (5 units)
    Nuclease-free H2O (NEB#B1500S) up to 100 µl
  2. Incubate at 37°C for 20 minutes.
  3. 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.


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RNase H