Poly(A) Polymerase Tailing Kit, 50 Rxns

Polyadenylate 3' ends of RNA molecules for various downstream applications

• Quality Kit: High enzymatic purity, specificity, and activity
• Flexible: Use polyadenylated RNAs in a variety of applications such as stabilizing in vitro transcribed RNA and adding primer (oligo-dT) binding sites to any RNA for first-strand cDNA synthesis
• Fast and Easy: Simple protocol produces polyadenylated RNA molecules quickly

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.

Applications

• 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

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.

Figure 1. Poly(A) tailing of RNA with the Poly(A) Polymerase Tailing Kit. The entire final product of an AmpliCap-Max™ in vitro transcription capping reaction (60 µg of a 1,760-base luciferase RNA) was treated with 8 units of Poly(A) Polymerase for the indicated times. For each sample, 0.1-µg aliquots were analyzed on a 1% agarose-formaldehyde gel.