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  • E. coli Poly(A) Polymerase

    Description

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

    Highlights

    • 3' labeling of RNA with ATP or Cordycepin
    • Preparing a priming site for cDNA synthesis using oligo-dT

    Product Source

    An E. coli strain that carries the cloned Poly(A) Polymerase gene from E. coli (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Adenosine-5'-Triphosphate (ATP)10 mM
    Poly(A) Polymerase Reaction Buffer10X

    Advantages and Features

    Applications

    • 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.

    Properties and 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
    Incubate at 37°C

    1X Poly(A) Polymerase Reaction Buffer:
    50 mM Tris-HCl
    250 mM NaCl
    10 mM MgCl2
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    300 mM NaCl
    1 mM DTT
    1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    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.

    Quality Control

    Quality Assurance Statement

    • Poly(A) Polymerase contains no detectable DNAses, RNAses and phosphatases. The purified protein contains no detectable DNA or RNA as determined by ethidium staining of an agarose gel.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    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?

    Selection Tools

    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.