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  • Labeling Protocol (M2080)

    Introduction

    This protocol is designed to label any RNA containing a 5´ triphosphate in a 20 µl reaction. The efficiency of label incorporation will be impacted by the molar ratio of RNA: GTP, as well as the ratio of cold and hot GTP. RNA used for labeling needs to be free of cold GTP and salts. Reaction size can be scaled up, as needed. The system provides enough reagents to perform 40 reactions at the 20 µl reaction scale.

    Protocol

    1. Combine RNA and Nuclease-free H2O in a 1.5 ml microfuge tube to a final volume of 14.0 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) 14.0 μl
      10X Capping Buffer 2.0 μl
      GTP mix (see notes on use #6, above) 2.0 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 labeled and 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.