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Exonuclease III

by Promega
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  • Overview
  • Protocols
  • Specifications
  • Resources
  • Exonuclease III catalyzes the stepwise removal of mononucleotides from dsDNA starting from a 3´-OH at nicks, blunt ends, recessed ends and 3´-overhangs of less than 4 bases, yielding nucleoside 5´-phosphates. This 3´?5´ exonuclease will also degrade DNA from 3´-phosphate ends due to its intrinsic 3´-phosphatase activity. In addition, the enzyme has apurinic endonuclease activity and ribonuclease H activity. The enzyme is used in conjunction with S1 nuclease for unidirectional deletion of sequences from the termini of DNA fragments. Exonuclease III is provided with a 10X Reaction Buffer consisting of 660mM Tris-HCl (pH 8.0 at 25°C), 6.6mM MgCl2.


    1. Weiss, B. (1976) J. Biol. Chem. 251, 1896–901.
    2. Rogers, S.G. and Weiss, B. (1980) Meth. Enzymol. 65, 201–11.
    3. Henikoff, S. (1984) Gene 28, 351–9.
    4. Promega Product Information #9PIM181, Promega Corporation.

    Storage Buffer: 20mM Tris-HCl (pH 7.5 at 25°C), 1mM DTT, 100mM KCl, 50% (v/v) glycerol.

    Source: Recombinant E. coli strain.

    QC Tests: Activity, SDS-PAGE/purity, endonuclease, 3´ overhang protection assay.

    Unit Definition: One unit is defined as the amount of enzyme required to produce 1nmol of acid-soluble nucleotides from double-stranded DNA in 30 minutes at 37°C.


    • Generating nested sets of deletions with double-stranded, linear DNA.
    • Preparation of single-stranded DNA.
  • Protocols

    Complete Protocol

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    Exonuclease III Protocol

    PDF (113 KB)

  • Certificate of Analysis

    Lookup Certificate of Analysis

    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

  • Resources