New England Biolabs, M0337S, Poly(U) Polymerase

Poly(U) Polymerase catalyzes the template independent addition of UMP from UTP or AMP from ATP to the 3´ end of RNA. Related Categories cDNA Synthesis & Reverse Transcriptases,, RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Applications PCR Specification Unit Definition One unit is defined as the amount of enzyme that incorporates 1 nmol of UMP into RNA in a 50 μl volume in 10 minutes at 37°C. Reaction Conditions 1X NEBuffer™ 2 Supplement with 0.5 mM UTP Incubate at 37°C 1X NEBuffer™ 2 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes Unit Assay Conditions 1X NEBuffer 2, 0.5 mM 3 H UTP and 5 µg yeast RNA are combined in a 50 μl reaction incubated at 37°C for 10 minutes. FAQ Q: What is the molecular weight of Poly(U) Polymerase? A: The molecular weight of Poly(U) Polymearse is 46 kDa. Q: What type of labeled-UTPs will the Poly(U) Polymerase incorporate? A: Poly(U) polymerase will incorporate radioactive nucleotides. Biotin or fluorophore-labeled nucleotides have not been tested. However, we predict that it will incorporate other labels, although at a reduced rate. Q: Will Poly(U) Polymerase incorporate GTPs onto the 3’ end of an RNA? A: Poly(U) polymerase can add a limited number (5 to 20) of G nucleotides to the 3' end of RNA. The length of poly G addition varies with different RNA substrates. In contrast, Poly(A) Polymerase is much more selective, exhibiting a strong preference for ATP compared to rGTP, rCTP or UTP. Few if any G's would be added with poly (A) polymerase. Q: What Poly(U) length can be expected to be incorporated by Poly(U) Polymerase? A: In the standard recommended reaction the tailing length for the Poly(U) polymerase is a function of both accessible primer and UTP concentration. Using a primer where 100% of the population is extended, a typical 30 minute incubation with 0.5 mM UTP will extend all RNA molecules approximately 1,000 bases. Q: Can Poly(U) Polymerase incorporate GTPs onto an RNA that contains chemically modified or 2-O-methyl groups on its 3’ end? A: Yes. However, Poly(U) Polymerase has a strong primer bias based upon the last few nucleotides on the 3’ end so in a mixed RNA population where some RNA may have modifications such as being double stranded or chemically modified with 2-O-methyl groups a longer incubation of the reaction may be required. Q: How can the Poly(U) Polymerase be inactivated without heating up the reaction? A: Poly(U) polymerase can be inactivated by adding EDTA to a final concentration of 20mM or higher. The tailed RNA can be purified by phenol-chloroform extraction or by column based procedures. Q: Does the E. coli Poly(U) Polymerase work in the M-MuLV reverse transcriptase buffer? A: Yes. RT buffer is compatible with Poly(U) polymerase. Q: Can Poly(U) Polymerase be used to add a poly U tail to ssDNA? A: No. Poly(U) polymerase requires an RNA primer.