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  • RNase Inhibitor, Murine

    Description

    RNase Inhibitor, Murine is a 50 kDa recombinant protein of murine origin. The inhibitor specifically inhibits RNases A, B and C. It inhibits RNases by binding noncovalently in a 1:1 ratio with high affinity. It is not effective against RNase 1, RNase T1, S1 Nuclease, RNase H or RNase from Aspergillus. In addition, no inhibition of polymerase activity is observed when RNase Inhibitor is used with Taq DNA Polymerase, AMV or M-MuLV Reverse Transcriptases, or Phage RNA Polymerases (SP6, T7, or T3).

    Recombinant murine RNase inhibitor does not contain the pair of cysteines identified in the human version that is very sensitive to oxidation, which causes inactivation of the inhibitor (1). As a result, RNase Inhibitor, Murine has significantly improved resistance to oxidation compared to the human/porcine RNase inhibitors, and is stable at low DTT concentrations (less than 1 mM). This makes it ideal for reactions where high concentration DTT is adverse to the reaction (eg. Real-time RT-PCR).

    Highlights

    • Improved resistance to oxidation, compared to human/porcine Rnase inhibitor 
    • Ideal for reactions where low DTT concentrations are required (e.g. Real-time PCR)

    Product Source

    An E. coli strain that carries the Ribonuclease Inhibitor gene from mouse.

    Advantages and Features

    Applications

    • RT-PCR
    • cDNA synthesis
    • In vitro transcription/translation
    • Enzymatic RNA labeling reaction
    • Other applications where the integrity of RNA is important

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of Murine RNase Inhibitor required to inhibit the activity of 5ng of RNase A by 50%. Activity is measured by the inhibition of hydrolysis of cytidine 2', 3'-cyclic monophosphate by RNase A.

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM HEPES-KOH
    50 mM KCl
    8 mM DTT
    50% Glycerol

    Quality Control

    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.
    • RNase Activity (Latent):
      The product is inactivated by heat treatment and tested in a reaction containing a RNA substrate. After incubationfor 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Since ribonucleases typically retain activity under denaturing conditions, care must be taken to avoid denaturing RNase Inhibitor molecules which have complexed with a ribonuclease. To prevent the release of active ribonuclease, temperatures greater than 50°C and high concentrations of urea or other denaturing agents should be avoided.
    2. The recommended concentration of RNase Inhibitor in a reaction is 1 unit/μl. During assembly of a reaction, RNase Inhibitor should be added before other components that are a possible source of RNase contamination (i.e. enzymes, plasmid from a mini prep.)

    References

    1. Kim, B.M. et al. Protein Science. 8, 430-434.

    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]

    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. Protocol for Avoiding Rnase Contamination using Murine Rnase Inhibitor (M0314)

    Selection Tools

    The inhibitor is a 50KD protein. It will be inactivated in conditions that will denature proteins.
    We recommend using the inhibitor at 1U/ul in your reaction.
    The inhibitor should be added in a reaction before other components that are a possible source of RNase contamination.
    The inhibitor should be used below 50?C.