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.
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 optimized 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
Unit Definition: One unit of Exonuclease III catalyzes 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.