Home Protocols 2´-O-Methylation of Capped RNA (M0366)

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

Introduction

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.

Protocol
  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.