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  • RNase HII

    recombinant unique buffer incubation temp heat inactivation no
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    M0288S250 units5,000 units/ml$67.00Add to Cart
    M0288L1,250 units5,000 units/ml$268.00Add to Cart
      
    Categories:
    RNases Products

    Description

    Ribonuclease HII (RNase HII) is an endoribonuclease that preferentially nicks 5´ to a ribonucleotide within the context of a DNA duplex. The enzyme leaves 5´ phosphate and 3´ hydroxyl ends (1). RNase HII will also nick at multiple sites along the RNA portion of an Okazaki fragment.

    Product Source

    An E. coli strain containing a genetic fusion of the RNase HII gene (rnhB) from E. coli and the gene coding for maltose binding protein (MBP). Following affinity chromatography, RNase HII is cleaved from the fusion construct by Factor Xa and then purified away from both MBP and Factor Xa. RNase HII cleaved from MBP has four additional amino acids at its N-terminus (Ile-Ser-Glu-Phe).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X

    Advantages and Features

    Applications

    Nicking of products generated with a polymerase that will incorporate ribonucleotides
    Generation of a double-stranded break at the site of an incorporated ribonucleotide when used with T7 Endo I
    Degradation of the RNA portion of Okazaki fragments

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to yield a fluorescence signal consistent with the nicking of 100 picomol of synthetic double-stranded DNA substrate containing a single ribonucleotide near the quencher of a fluorophore/quencher pair in 30 minutes at 37°C in 1X ThermoPol Buffer.

    Reaction Conditions

    1X ThermoPol® Reaction Buffer
    Incubate at 37°C

    1X ThermoPol® Reaction Buffer:
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton® X-100
    pH 8.8 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    1 mM EDTA
    50% Glycerol

    Heat Inactivation

    No

    Unit Assay Conditions

    1X ThermoPol Reaction Buffer with 30 nM of a synthetic dsDNA 26-mer containing an internal ribonucleotide in a total reaction volume of 150 µl.

    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.

    Notes

    1. RNase HII displays reduced hybridase activity compared to RNase H (2).
    2. Okazaki fragments are preferentially nicked 5´ to the ribonucleotide at the junction of the RNA/DNA sequence (1).
    3. RNase HII prefers a dsDNA duplex containing a single ribonucleotide over a RNA/DNA hybrid substrate (1) or an Okazaki fragment (3).
    4. Incubation with 0.1% SDS is sufficient to inactivate RNase HII.

    References

    1. Rydberg, B. and Game, J. (2002). Proc. Natl. Acad. Sci.. 99
    2. Itaya, M. (1990). Proc. Natl. Acad. Sci.. 87
    3. Nichols, N.M. Unpublished Observation.

    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. RNase HII cannot be heat-inactivated. How can RNase HII be inactivated?
    2. Which side of the ribonucleotide does RNase HII cut?
    1. Reaction Protocol for RNase HII (M0288)