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Product description: Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3'-hydroxyl termini of RNA molecules.
Source: Recombinant E.coli
Concentration and Size: 5U/μL
Unit Definition: One unit is defined as the amount of enzyme that will incorporate 1nmol of AMP into RNA in a 20μL volume in 10 minutes at 37°C.
Storage Buffer: 20mM Tris-HCl (pH7.5@25℃), 1mM EDTA, 300mM NaCl, 1mM DTT, 0.1%(v/v) Triton X-100,50%(v/v) glycerol.
Companion Product: 10X poly(A) Polymerase Reaction Buffer, Cat#ON-127, 500mM Tris-HCl, (pH7.9@25℃), 2.5M NaCl, 100mM MgCl2.
Protocol: The standard reaction will produce ～150bp long poly(A)-tail on up to 10ug of capped or uncapped RNA from IVT reactions and co-transcriptional capping reactions need to be purified before adding to the poly(A)-tailing reaction.
1. Combine the following reagents:
|10X poly(A) Polymerase Reaction Buffer||2μL|
|(Optional) RNase inhibitor||20units|
|Nuclease-free H2O||to 20μL|
2. Incubate reaction at 37°C for 60 minutes.
3. Stop reaction by adding EDTA to final concentration of >11mM.
* Before poly(A)-tail reaction, heat-denaturation of the RNA may improve the efficiency of adding poly(A)-tail.
Storage Conditions: -20℃
Quality Assurance: Free of endonuclease, exonuclease and RNase activities.
Physical Purity: ＞95% by SDS-PAGE.
1. Bernstein P and Ross J (1989) Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem Sci 14: 373–377.
2. Gallie DR (1991) The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev 5: 2108–2116.
3. Krug, M.S. and Berger, S.L. (1987) Methods Enzymol. 152, 262.