Poly (A) tailing reaction

The addition of a poly (A) tail was performed using E. coli Poly (A) polymerase (New England Biolabs, cat. no. M276S), following manufacturer's instructions. Briefly, up to 10 μg of capped RNA was diluted to 15 μl using nuclease-free water. To the RNA, 2 μl of 10× E. coli Poly (A) polymerase reaction buffer, 2 μl of 10-mM ATP and 1 μl of E. coli Poly (A) polymerase (5 units) were added and incubated for 30–60 min at 37°C. The capped and tailed RNA was purified using the RNA cleanup protocol described earlier and quantitated. Analysis of the synthetic mRNA by agarose gel electrophoresis demonstrated that the efficiency of poly (A) tail addition was >95% (Figure 1).

From left to right, the 1-kb ladder and the indicated synthetic RNAs before (-) and after (+) enzymatic addition of the poly (A) tail.

eGFP: Enhanced green fluorescent protein.