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  • Exonuclease V (RecBCD)

    Description

    Exonuclease V, a RecBCD complex from E. coli has several different enzyme activities, including an ATP-dependent single-stranded DNA endonuclease activity, ss- and ds- DNA exonuclease activity. The hydrolysis in each case is bi-directional (from both the 3´ and 5´ ends) and processive, producing oligonucleotides (1,2,3). All Exonuclease V activities have divalent cation requirements. Mg2+ is required for the exonuclease activity, while Ca2+ inhibits the exonuclease activity and allows double-stranded DNA unwinding (helicase activity) without hydrolysis (4,5).

    Product Source

    An E. coli strain containing plasmids for expressing the three subunits of E. coli Exonuclease V: RecB, RecC and RecD.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X
    Adenosine 5'-Triphosphate (ATP)-20*10 mM

    * Reagents' Storage Notes

    • Adenosine 5'-Triphosphate (ATP):

      For long term storage (>30 days), store at -70°C.

    Advantages and Features

    Applications

    Degradation of linear ssDNA and dsDNA while preserving nicked and supercoiled plasmid DNA.

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to produce 1 nmol of acidsoluble deoxyribonucleotide from double-stranded DNA in 30 minutes at 37°C in a total reaction volume of 50 μl.

    Reaction Conditions

    1X NEBuffer 4
    Supplement with 1 mM ATP
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    70°C for 30 min

    Unit Assay Conditions

    1X NEBuffer 4, 1 mM ATP with 0.15 mM sonicated duplex [3H]-DNA.

    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 (Nicked Double Stranded DNA):
      The product is tested in a reaction containing a nicked DNA substrate. After incubation for 4 hours the percent cleaved is determined by agarose gel electrophoresis.
    • 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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • 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.

    References

    1. Eichler, D.C. et al. (1977). J.Biol. Chem. 252, 499-503.
    2. Tayler, A.F. et al. (1985). J. Mol. Biol. 185, 431-443.
    3. Amundsen, S.K. et al. (1986). Proc. Natl. Acad. Sci. 83, 5558-5562.
    4. Palas, K.M. et al. (1990). J. Biol. Chem. 265, 3447-3454.
    5. Dillingham, M.S. et al. (2008). Microbiology and Mol. Biol. Review. 72, 642-671.

    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. A Typical Exonuclease V Reaction (M0345)

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