Poly(A) Polymerase Tailing Kit

• Addition of a poly(A) tail to RNA synthesized in vitro in order to increase the stability of the RNA and enhance its ability to be translated in vivo after transfection or microinjection into eukaryotic cells.^1-3
• Addition of a poly(A) tail to an RNA molecule or a mixture of RNA molecules in order to provide a priming site for synthesis of first-strand cDNA using a primer with poly(dT) on its 3´ end.
• Synthesis of polyadenylated RNA for nucleic acid amplification methods or gene expression studies.
• 3´-end labeling of RNA with radioactive A residues.⁴
• Quantifying mRNA.⁵

Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3´-hydroxyl termini of RNA molecules.⁶ The Poly(A) Polymerase Tailing Kit provides the enzyme and other reagents for quickly and easily adding a poly(A) tail to the 3´ end of any RNA. Poly(A) Polymerase is encoded by an E. coli gene that has been cloned into a plasmid and overexpressed in an E. coli strain.

Benefits

• High enzymatic purity, specificity, and activity.
• Easy, rapid, and highly efficient poly(A) tailing of RNA (Fig. 1).

Unit Definition: One unit of Poly(A) Polymerase catalyzes the incorporation of 1 nmol of AMP into acid-insoluble form in 10 minutes at 37°C under standard assay conditions.

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton® X-100.

10X Reaction Buffer: 0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, and 100 mM MgCl₂. A separate 10-mM ATP Solution is also provided.

Quality Control: Poly(A) Polymerase is tested for polyadenylation of RNA in vitro. It is free of detectable exo- and endonuclease and RNase activity.

References

1. Drummond, D.R. et al. (1985) J. Cell. Biol. 100, 1148.
2. Galili, G. et al. (1988) J. Biol. Chem. 263, 5764.
3. Belasco, J. and Brawerman, G. (1993) Control of Messenger RNA Stability, Academic Press, San Diego, CA.
4. Lingner, J. and Keller, W. (1993) Nucleic Acids Res. 21, 2917.
5. Krug, M.S. and Berger, S.L. (1987) Methods Enzymol. 152, 262.
6. Gething, M. et al. (1980) Nature 287, 301.

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