NEB, M0276L, E. coli Poly(A) Polymerase

[{"group": "General", "name": "Materials Required but not Supplied Nuclease-free Water (NEB #B1500) RNase Inhibitor, Murine (NEB #M0314) (optional) Unit Definition One unit is defined as the amount of enzyme that will incorporate 1 nmol of AMP into RNA in a 20 μl volume in 10 minutes at 37°C. Reaction Conditions 1X Poly(A) Polymerase Reaction Buffer Supplement with 1 mM ATP Incubate at 37°C 1X Poly(A) Polymerase Reaction Buffer 50 mM Tris-HCl 250 mM NaCl 10 mM MgCl2 (pH 8.1 @ 25°C) Storage Buffer 20 mM Tris-HCl 300 mM NaCl 1 mM DTT 1 mM EDTA 50% Glycerol 0.1% (w/v) Triton® X-100 pH 7.5 @ 25°C Unit Assay Conditions 1X Poly(A) Polymerase Reaction Buffer, 1 mM rATP and 500 ng 5´ FAM labeled poly A 20-mer RNA in a 20 μl reaction. After incubation at 37°C 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.", "value": ""}]