Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA. Isolated from a recombinant source. Source: An E. coli strain that carries the cloned Poly(A) Polymerase gene from E. coli. Applications: RT-qPCR, RT-PCR and cDNA Synthesis, PCR Labeling of RNA with ATP or cordycepin Poly(A) tailing of RNA for cloning or affinity purification Enhances translation of RNA transferred into eukaryotic cells. Unit Definition: One unit is defined as the amount of enzyme that will incorporate 1nmol of AMP into RNA in a 20ul volume in 10 minutes at 37C. Reaction Conditions: 1X Poly(A) Polymerase Reaction Buffer Supplement with 1mM ATP Incubate at 37C 1X Poly(A) Polymerase Reaction Buffer: 50mM Tris-HCl 250mM NaCl 10mM MgCl2 (pH 8.1 25C) Storage Buffer: 20mM Tris-HCl 300mM NaCl 1mM DTT 1mM EDTA 50% Glycerol: 0.1% (w/v) Triton X-100 pH 7.5 25C Unit Assay Conditions: 1X Poly(A) Polymerase Reaction Buffer, 1mM rATP and 500ng 5´ FAM labeled poly A 20-mer RNA in a 20ul reaction. After incubation at 37C for 10 minutes the length of the poly(A) addition is determined either by gel electrophoresis or with an automated capillary DNA sequencer. In this assay 5 units of enzyme add approximatley 60 to 80 adenosines to the RNA primer. In these conditons 20 units of enzyme will deplete the rATP.
Form:
rATP is not included in the buffer supplied.
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