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  • T5 Exonuclease


    T5 Exonuclease degrades DNA in the 5´ to 3´ direction (1). T5 Exonuclease is able to initiate nucleotide removal from the 5´ termini or at gaps and nicks of linear or circular dsDNA (1). However, the enzyme does not degrade supercoiled dsDNA (2). T5 Exonuclease also has ssDNA endonucleae activity.

    Product Source

    An E. coli strain that carries a plasmid with the T5 phage D15 gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X

    Advantages and Features


    • Degradation of linear ssDNA, dsDNA or nicked plasmid DNA while maintaining supercoiled plasmid DNA.
    • Remove denatured DNA isolated during alkaline lysis-based plasmid purification procedure (5).
    • Enhance the transfection efficiency of miniprep from plasmid cDNA libraries (6).

    Properties and Usage

    Unit Definition

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

    Reaction Conditions

    1X NEBuffer 4
    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


    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


    Unit Assay Conditions

    1X NEBuffer 4, 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 (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.


    1. Sayers, J.R. et al. (1991). Nucleic Acids Res. 19, 4127-4132.
    2. Sayers, J.R. et al. (1990). J. of Biol. Chem. 265, 18311-18317.
    3. Kaliman, A.V. et al. (1986). FEBS. 195, 61-64.
    4. Frenkel, G.D. et al. (1971). J. of Biol. Chem. 246, 4839-4847.
    5. Sayers, J.R. et al. (1996). Analytical Biochem. 241, 186-189.
    6. Kiss-Toth, E. et al. (2001). Analytical Biochem. 288, 230-232.

    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.


    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. How Does T5 Exonuclease differ from Lambda Exonuclease (NEB# M0262)?
    2. Will T5 exonuclease cleave supercoiled dsDNA?
    3. How does T5 Exonuclease differ from Exonuclease III (NEB# M0206)?
    4. What is the activity of T5 Exonuclease in the NEBuffers?
    5. Can T5 Exonuclease be heat inactivated?
    6. Can DNA be blunted using T5 Exonuclease?
    1. Protocol for T5 Exonuclease (M0363)

    Selection Tools

    Has 5’ to 3’ dsDNA exonuclease activity and ssDNA endonuclease activity.