Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)

This protocol is designed to tail up to 10 µg of RNA in a 20 µl reaction. Reaction size can be scaled up as needed. In a poly (A) tailing reaction, the length of the tail depends on the molar concentration of the RNA 3'OH ends, reaction time, amount of enzyme and ATP concentration. Tail length can be modified by changing one or more of these factors. As a general guideline, incubation of 5 units of the enzyme with 1-10 µg RNA in a 20 µl reaction at 37°C for 30 minutes (with 1X Reaction buffer and 1mM ATP) will result in a tail length of greater than 100 bases.
  • RNA used for tailing reactions should be purified prior to use and suspended in nuclease-free water. EDTA should not be present and the solution should be free of salts.
  • RNase Inhibitor may be added to enhance the stability of the RNA in solution. If this is desired, 0.5 µl of a standard RNase inhibitor (such as RNase Inhibitor, Murine, NEB #M0314) can be added at the time of reaction set-up. The additional volume can be subtracted from the amount of H2O used in the reaction.
  1. Add the following components in the order specified:

  2. Component Volume
    RNA 1-10 µg in 15 µl nuclease free water
    10X E. coli Poly(A) Polymerase Reaction Buffer 2 µl (1X)
    ATP (10mM) 2 µl
    E. coli Poly(A) Polymerase 1 µl
    Total 20 µl

  3. Incubate reaction at 37°C for 30 minutes.
  4. Stop the reaction by adding EDTA to a final concentration of 10 mM or directly proceeding to the cleanup step.