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  • Poly(U) Polymerase

    Description

    Poly(U) Polymerase catalyzes the template independent addition of UMP from UTP or AMP from ATP to the 3´ end of RNA.

    Product Source

    An E. coli strain that carries the cloned poly(U) polymerase gene of Schizosaccharomyces pombe Cid1.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Advantages and Features

    Applications

    • Labeling of RNA with UTP
    • Poly(U) and Poly(A) tailing of RNA for cloning
    • Studying effects of poly(U) tailing on stability and translation of RNA transferred into eukaryotic cells
    • Poly (A) tailing of 2' O-Me modified 3' ends

    Properties and Usage

    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 1 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 Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Unit Assay Conditions

    1X NEBuffer 2, 0.5 mM 3H UTP and 5 µg yeast RNA are combined in a 50 µl reaction incubated at 37°C for 10 minutes.

    Quality Control

    Quality Assurance Statement

    • Poly(U) Polymerase contains no detectable DNAses and RNAses. The purified protein contains no detectable DNA or RNA.

    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. UTP is not included with this product.
    2. Poly(U) Polymerase in NEBuffer 2 will incorporate rNMP from rNTP into RNA. Tailing length of poly(U) varies with UTP. Poly(U) Polymerase is highly processive under low primer concentrations (<100 pmol).

    References

    1. Wickens, M. and Kwak, J.E. (2008). Science. 319, 1344.
    2. Kwak, J.E. and Wickens, M. (2008). RNA. 13, 860.
    3. Rissland, O.O.S., Mikulasova, A. and Norbury, C.J. (2007). Molecular and Cell Biology. 27, 3612.

    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. What is the molecular weight of Poly(U) Polymerase?
    2. What type of labeled-UTPs will the Poly(U) Polymerase incorporate?
    3. Will Poly(U) Polymerase incorporate GTPs onto the 3’ end of an RNA?
    4. What Poly(U) length can be expected to be incorporated by Poly(U) Polymerase?
    5. Can Poly(U) Polymerase incorporate GTPs onto an RNA that contains chemically modified or 2-O-methyl groups on its 3’ end?
    6. How can the Poly(U) Polymerase be inactivated without heating up the reaction?
    7. Does the E. coli Poly(U) Polymerase work in the M-MuLV reverse transcriptase buffer?
    8. Can Poly(U) Polymerase be used to add a poly U tail to ssDNA?
    1. A Typical Tailing Reaction (M0337)

    Selection Tools

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