Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3'-hydroxyl termini of RNA molecules.1 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 in a plasmid and overexpressed in an E. coli strain. 

Unit Definition

One unit of Poly(A) Polymerase catalyzes the incorporation of 1 nanomole of AMP into acid-insoluble form in 10 minutes at 37°C in a reaction mixture consisting of 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10 mM MgCl2, and 1 mM ATP. 

Storage Buffer

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

10X Poly(A) Reaction Buffer

0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, and 100mMMgCl2. A separate 10mM 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.

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Protocols for: Poly(A) Polymerase Tailing Kit

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Epicentre’s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

Poly(A) Polymerase Tailing Kit Protocol

(catalogue number PAP5104H)

Please note: all protocols off site are the responsibility of the products supplier

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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, 30.
7. Carbery, I. D., et al. (2010) Targeted Genome Modification in Mice Using Zinc Finger Nucleases. Genetics  , genetics.110.117002.
8. Carbery, I. D., et al. (2010) Targeted Genome Modification in Mice Using Zinc-Finger Nucleases. Genetics 186 , 451-459.
9. Leifer, I., et al. (2010) Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany. J Gen Virol 91 , 2687-2697.
10. Marchese, F. P., et al. (2010) MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment. J Biol Chem 285 , 27590-27600.
11. Rederstorff, M., et al. (2010) RNPomics: Defining the ncRNA transcriptome by cDNA library generation from ribonucleo-protein particles. Nucleic Acids Res , gkq057.

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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

Benefits

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

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200