Removal of pyrophosphate from 5’ triphosphorylated RNA using RppH (NEB #M0356)

1.    Set up the reaction as shown below-
 

Component	Volume
5’ triphosphorylated RNA	Up to 2 µg
NEBuffer 2	2 µl
RNA 5’ Pyrophosphohydrolase (RppH)	2 µl
Nuclease-free water	to 20 µl

 
2.    Incubate at 37°C for 30 minutes.

3.    Stop the reaction by adding EDTA to a final concentration of 20mM. Alternatively, you can directly proceed to RNA clean up.
 
Note: The EDTA chelates Mg++ ions so if you intend to follow the RppH reaction with another enzymatic reaction that uses Mg++ without reaction clean up, avoid adding EDTA. We recommend purifying RNA between different enzymatic steps to ensure optimal performance of the enzymes.