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  • 2´-O-Methylation of Capped RNA (M0366)


    This protocol is designed to methylate up to 10 μg of capped RNA in a 20 μl reaction. Reaction size can be scaled up as needed.


    1. Combine capped RNA and nuclease-free water in a final volume of 16 μl. (Refer to step 1 in the notes on use).

    2. Heat at 65°C for 5 minutes (Refer to step 2 in the notes on use).

    3. Place tube on ice for 5 minutes.

    4. Add the following components in the order specified:
      Denatured capped RNA (from above) 16.0 μl
      10X Capping Buffer 2.0 μl
      SAM (4 mM, dilute 32 mM stock to 4 mM) 1.0 μl
      mRNA Cap 2´-O-Methyltransferase (50 U/μl) 1.0 μl
        20.0 μl

      Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor (e.g., Murine RNase Inhibitor NEB #M0314 ) during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.

    5. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).

    6. Proceed with purification of the RNA (if required) for downstream applications.