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  • Capping Protocol (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.