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  • Pyrophosphatase, Inorganic (E. coli)

    Description

    Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate:

    Product Source

    PPase is prepared from an E. coli strain containing a clone of the E. coli inorganic pyrophosphatase gene.

    Advantages and Features

    Applications

    • Increasing RNA yield in transcription reaction; enhancing DNA replication.

    Properties and Usage

    Unit Definition

    One unit is the amount of enzyme that will generate 1 µmol of phosphate per minute from inorganic pyrophosphate under standard reaction conditions (a 10 minute reaction at 25°C in 20 mM Tris-HCl, pH 8.0, 2 mM MgCl2 and 2 mM  PPi).

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 8.0 @ 25°C

    Heat Inactivation

    No

    Quality Control

    Quality Assurance Statement

    • This enzyme is validated in an in vitro RNA synthesis reaction.

    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.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • 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. Use 1–3 units per ml in a high yield in vitro RNA synthesis reaction.

    Supporting Documents

    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]

    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.
    The enzyme works well in most reaction buffers containing Mg2+.
    It cannot be inactivated by heating at 70°C.