Product Class: Other

E. coli Poly(A) Polymerase
NEBU recombinant 37

Product Introduction

Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA

  • Isolated from a recombinant source
  • Applications include 3' labeling of RNA with ATP or Cordycepin and poly(A) tailing of RNA for cloning or affinity purification
  • Enhances translation of RNA transferred into eukaryotic cells
  • Tested for the absence of DNases and RNases
Catalog # Size Concentration
M0276S 100.0 units 5000 units/ml
M0276L 500.0 units 5000 units/ml

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Product Information

Description

Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA.

Figure 1. Analysis of capped and polyadenylated RNA
Figure 1
A. Agilent Bioanalyzer® analysis of capped and polyadenylated RNA. Longer tails are produced by increasing the enzyme concentration in the reaction. Calculated A-tail lengths are indicated over each lane. Lanes: L: size marker,1: No poly-A tail, 2: 5 units, 3 :15 units, 4 : 25 units of E. coli Poly(A) Polymerase. B. Effect of enzymatic A-tailing on the luciferase reporter activity of CLuc mRNA

Product Source

An E. coli strain that carries the cloned Poly(A) Polymerase gene from E. coli (1).
This product is related to the following categories:
RNA Modification,
cDNA Synthesis & Reverse Transcriptases Products,
RNA Synthesis In vitro Transcription (IVT),
This product can be used in the following applications:
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0276S     -20    
      E. coli Poly(A) Polymerase M0276SVIAL -20 1 x 0.02 ml 5,000 units/ml
      Poly(A) Polymerase Reaction Buffer B0276SVIAL -20 1 x 1.5 ml 10 X
      Adenosine-5'-Triphosphate (ATP) B0756AVIAL -20 1 x 0.2 ml 10 mM
  • M0276L     -20    
      E. coli Poly(A) Polymerase M0276LVIAL -20 1 x 0.1 ml 5,000 units/ml
      Poly(A) Polymerase Reaction Buffer B0276SVIAL -20 1 x 1.5 ml 10 X
      Adenosine-5'-Triphosphate (ATP) B0756AVIAL -20 1 x 0.2 ml 10 mM

Properties & Usage

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.

Application Features

  • 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.

Product Notes

  1. rATP is not included in the buffer supplied.

References

  1. Cao, G.J. and Sarkar, N. (1992). Proc.Natl. Acad. Sci. USA. 89, 10380-10384.

Protocols, Manuals & Usage

Protocols

  1. Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How can the E. coli Poly(A) Polymerase be inactivated without heating up the reaction?
  2. Does the E. coli Poly(A) Polymerase work in the M-MuLV reverse transcriptase buffer?
  3. Can E. coli Poly(A) Polymerase be used to add a poly A tail to ssDNA?
  4. Can E. coli Poly(A) Polymerase also add GTP, UTP and CTP to RNA?
  5. Can I end-label a polyadenylated RNA molecule with a Cy3/5 or biotin-modified base using the E. coli Poly(A) Polymerase?
  6. Does E. coli Poly(A) Polymerase work on tRNA?
  7. Is MnCl2 required for a reaction with E. coli Poly(A) Polymerase?
  8. Is it possible to have the same length of poly A added to all RNA molecules by E. coli Poly(A) Polymerase?
  9. 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?
  10. 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?
  11. What is the molecular weight of E.coli Poly(A) polymerase?

Tech Tips

E. coli Poly (A) Polymerase (M0276)

Vortex tube gently before use.

Poly(A) Polymerase can be inactivated by adding EDTA at a final concentration of 10 mM to the reaction.

Poly (A) Polymerase is compatible with the M-MuLV reverse transcriptase buffer. If the intention is to carry out the polyadenylation step followed by reverse transcription, we recommend to set-up these reactions in two sequential steps. The polyadenylation reaction can be done first using the reverse transcriptase buffer. This can be followed by addition of the components necessary for reverse transcription.

We strongly recommend wearing gloves, using nuclease-free tubes and reagents, and thoroughly cleaning pipettes and bench surfaces to avoid RNase contamination.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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