New England Biolabs, M0276L, E. coli Poly(A) Polymerase

Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA Related Categories RNA Modification,, cDNA Synthesis & Reverse Transcriptases,, RNA Synthesis In vitro Transcription (IVT), Applications PCR Specification 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. FAQ Q: How can the E. coli Poly(A) Polymerase be inactivated without heating up the reaction? A: Poly(A) Polymerase can be inactivated by adding EDTA at a final concentration of 10 mM into the reaction buffer. Q: Does the E. coli Poly(A) Polymerase work in the M-MuLV reverse transcriptase buffer? A: Yes. Poly(A) polymerase works well in the M-MuLV reverse transcriptase buffer. If what is intended is to carry out the polyadenylation step and then the RT reaction, we recommend to set-up these reactions in two sequential steps. The polyadenylation reaction can be done first using the reverse transcriptase buffer, then the additional components of the RT can be added to carry out the reverse transcription reaction. Q: Can E. coli Poly(A) Polymerase be used to add a poly A tail to ssDNA? A: No. E. coli poly(A) polymerase will not add A's to single-stranded DNA. However, Terminal Transferase (M0315) is a DNA polymerase that will add a poly dA tail to the 3' end of single stranded DNA using dATP. Please note that Terminal Transferase will not use ATP. Q: Can E. coli Poly(A) Polymerase also add GTP, UTP and CTP to RNA? A: Poly (A) Polymerase is very selective for the addition of ATP to the 3' end of RNA. However, Poly (U) Polymerase (M0337) will add all four ribo NTPs to RNA. The enzyme is most active with UTP, but it will also add other NTPs at slower rates. However, please note, that Poly (U) Polymerase can exhibit some activity bias based on the 3’ sequence of the RNA. In some of those instances the enzyme will not add the alternative NTPs efficiently. Q: Can I end-label a polyadenylated RNA molecule with a Cy3/5 or biotin-modified base using the E. coli Poly(A) Polymerase? A: Yes. Biotin-ATP can be incorporated efficiently by the Poly (A) Polymerase. Cy3/5 modified bases can also be incorporated, but at a lower efficiency. The tailing length and biotin density can be controlled by optimizing the reaction time, the ATP/biotin-ATP ratio and the total ATP concentration. Q: Does E. coli Poly(A) Polymerase work on tRNA? A: Yes. The E. coli Poly(A) Polymerase works on tRNA as long as the tRNA has a free 3’ end. Q: Is MnCl2 required for a reaction with E. coli Poly(A) Polymerase? A: The enzyme requires the presence of a divalent cation for activity. In the absence of MgCl2, MnCl2 can be added at a concentration not higher than 1 mM. Q: Is it possible to have the same length of poly A added to all RNA molecules by E. coli Poly(A) Polymerase? A: No. The number of As added to a pool of RNA molecules has a Gaussian distribution, so it is not possible to plan to add a certain amount of A's to all RNA molecules present in a reaction. Q: Can we use the ribonucleotide Mix (N0466) to replace the addition of an rATP solution for the poly A tailing of an RNA template by E.coli Poly(A) polymerase? A: The use of the rNTP mix in an E.coli Poly(A) polymerase reaction is not recommended because nucleotides other than ATP will not be incorporated by the Poly(A) polymerase and they potentially can inhibit the reaction. We sell rATP as a separate product (P0756S). Q: Is the E.coli Poly(A) polymerase able to elongate short and surface-immobilized oligoribonucleotides? What is the minimum length of an RNA sequence that can be recognized by the enzyme and, therefore, elongated? A: The E. coli poly (A) polymerase can add poly As to a 20 base long RNA oligo. We have not tried to add poly A to a bound RNA oligo. Q: What is the molecular weight of E.coli Poly(A) polymerase? A: The molecular weight of E. coli Poly(A) Polymerase is 54.6kDa.