Cap-0 synthesis using Vaccinia Capping Enzyme (NEB #M2080)

Introduction

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.

Protocol

1. Combine RNA and Nuclease-free H₂O in a 1.5 ml microfuge tube to a final volume of 15.0 µl.
   
   
2. Heat at 65°C for 5 minutes.
   
   
3. Place tube on ice for 5 minutes.
   
   
4. Add the following components in the order specified:

   Denatured RNA (from above)	15.0 µl
   10X Capping Buffer	2.0 µl
   GTP (10 mM)	1.0 µl
   SAM (2 mM, dilute 32 mM stock to 2 mM)	1.0 µl
   Vaccinia Capping Enzyme	1.0 µl
   Total Volume	20 µl

5. Incubate at 37°C for 30 minutes.
   
   
6. 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.