This protocol is designed to cap up to 10 µg of RNA (100 nt or larger) in a 20 µl reaction. Reaction size can be scaled up, as needed. The system provides enough reagents to perform 40 reactions at the 10 µg RNA/20 µl reaction scale.
- Combine RNA and Nuclease-free H2O in a 1.5 ml microfuge tube to a final volume of 15.0 µl.
- Heat at 65°C for 5 minutes.
- Place tube on ice for 5 minutes.
- Add the following components in the order specified:
|Denatured RNA (from above)
|10X Capping Buffer
|GTP (10 mM)
|SAM (2 mM, dilute 32 mM stock to 2 mM)
|Vaccinia Capping Enzyme
- Incubate at 37°C for 30 minutes.
- RNA is now capped and ready for use in downstream applications. Some applications may require RNA to be purified prior to use. If the RNA needs a poly(A) tail, NEB Poly(A) Polymerase (NEB #M0276 ) can be used.