One-Step Capping and 2´-O-Methylation (M0366)
IntroductionThis protocol is designed to complete both capping and 2´-O-methylation in a single step. It involves incubating uncapped RNA with the Vaccinia Capping Enzyme (NEB #M2080, not included) and mRNA Cap 2´-O-Methyltransferase in the presence of GTP and SAM. The Vaccinia Capping Enzyme adds the cap at the 5´ end of the RNA followed by 2´-Omethylation by the methyltransferase. This protocol can synthesize up to 10 μg of cap-1 RNA in a 20 μl reaction. Reaction size can be scaled up as needed.
Combine uncapped RNA and nuclease-free
water in a final volume of 14.0 μl. (Refer to step 1 in the notes on
- Heat at 65°C for 5 minutes (Refer to step
2 in the notes on use).
- Place tube on ice for 5
- Add the following components in the order
Denatured uncapped RNA (from above) 14.0 μl 10X Capping Buffer 2.0 μl GTP (10 mM) 1.0 μl SAM (4 mM, dilute 32 mM stock to 4 mM) 1.0 μl Vaccinia Capping Enzyme (10 U/μl) 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.
- Incubate at 37°C for 60 minutes (For RNA
less than 200 nt long increase incubation time to 2 hours).
- Proceed with purification of the RNA (if required) for downstream applications.