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Exonuclease III

This product includes both Exonuclease III enzyme and a 10X reaction buffer.

Exonuclease III

Digest duplex DNA in a 3´ to 5´ direction from a nick, blunt end, or 3´-recessed end, producing stretches of ssDNA.

Key features

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  • Produce stretches of ssDNA in dsDNA templates containing nicks, blunt ends or recessed 3' ends
  • Protect protruding 3' ends because this exonuclease will not start digestion at protruding 3' overhangs

Size: 25000 units

Item ID EX4425K
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Product information

Exonuclease III digests duplex DNA in a 3´ to ;5´ direction from a nick, a blunt end, or 3´-recessed end, producing stretches of ssDNA on the opposite strand. 5,6 Under defined reaction conditions, DNA degradation by Exonuclease III proceeds at a uniform rate yielding predictable and reproducible digestion results.


  • Production of intermediates for site-directed mutagenesis. 1-3
  • Production of strand-specific radiolabelled probes. 4

Because the rate of exonucleolytic excision of deoxyribonucleotides by Exonuclease III is dependent upon reaction factors including temperature, ionic strength, template sequence, and enzyme-to-DNA ratios, 4,7 each template must be optimised using sample digestions to achieve the desired excision rate.

Exonuclease III is not active on 3´-protruding ends of four bases or more in length, ssDNA, or on thioester-linked nucleotides. 2 The enzyme also has intrinsic RNase H, 3´-DNA phosphatase, and apurinic DNA endonuclease activities. 1,6


  1. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
  2. Vandeyar, M.A. (1988) Gene 65, 129.
  3. Luckow, B. et al. (1987) Nucleic Acids Res. 15, 417.
  4. Richardson, C.C. et al. (1964) J. Biol. Chem. 239, 251.
  5. Weiss, B. (1976) J. Biol. Chem. 251, 1896.
  6. Rogers, S.G. and Weiss, B. (1980) Meth. Enzymol. 65, 201.
  7. Guo, L.H. and Wu, R. (1982) Nucleic Acids Res. 10, 2065.

Unit Definition: One unit of Exonuclease III catalyses the release of 1 nmol of acid-soluble nucleotides from double stranded calf thymus DNA in 30 minutes at 37 °C under standard assay conditions.

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.

Exonuclease III 10X Reaction Buffer: 330 mM Tris-acetate (pH 7.5), 660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT.

Quality Control: Exonuclease III is free of detectable exogenous RNase, endonuclease, and single-stranded exonuclease activities.

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