Exonuclease III digests duplex DNA in a 3'Æ5' direction from a blunt end, 5'-overhang or nick, producing stretches of single-stranded DNA. Under defined reaction conditions, DNA degradation by Exonuclease III proceeds at a uniform rate yielding predictable and reproducible 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,3,4 each template must be optimised using sample digestions to achieve the desired excision rate.
Exonuclease III is not active on 3'-protruding ends of 4 bases or more in length, single-stranded DNA, or on thioester-linked nucleotides. The enzyme also has intrinsic RNase H, 3'-DNA phosphatase, and apurinic DNA endonuclease activities.
One unit catalyses the release of 1nmole of acid-soluble nucleotides from double-stranded calf thymus DNA in 30 minutes at 37°C in 33mM Tris-acetate, pH 7.8, 66mM potassium acetate, 10mM magnesium acetate, and 0.5mM DTT.
50% glycerol containing 50 mM Tris-HCl, pH 7.5, 0.1M NaCl, 0.1mM EDTA, 1mM DTT, and 0.1% Triton® X-100.
Exonuclease III 10X Reaction Buffer
330mM Tris-acetate, pH 7.8, 660mM potassium acetate, 100mM magnesium acetate, and 5mM DTT.
Exonuclease III is free of detectable exogenous RNase, endonuclease, and single-stranded exonuclease activities.
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