Exonuclease I (Exo I) digests single-stranded DNA in a 3'→5' direction. Although it requires the presence of magnesium and a free 3'-hydroxyl terminus for activity, Exo I is active under a wide variety of buffer conditions and can be added directly into most reaction mixes. Exo I can be heat-inactivated by incubation at 80°C for 15 minutes.
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Figure 1. Specificity of Exonuclease I for ssDNA. 200 ng of EcoR I-linearized pUC19 dsDNA and 1 µg of a 100-mer ssDNA oligo were mixed in 1X TA Buffer and incubated at 37°C for 20 min in the absence or presence of 10 units of Exonuclease I. As seen on this 1% agarose gel, Exonuclease I completely digested the linear ssDNA oligo, but left the linearized plasmid dsDNA intact. Lane 1, size markers; Lane 2, minus Exo I; Lane 3, plus Exo I.
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Unit Definition
One unit catalyses the release of 10nmoles of acid-soluble nucleotides from heat-denatured calf thymus DNA in 30 minutes at 37°C in 33 mM Tris-acetate, pH 7.8, 66mM potassium acetate, 10mM magnesium acetate, and 0.5mM DTT.
Storage Buffer
50% glycerol containing 50mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.1mM EDTA, 1mM DTT, and 0.1% Triton® X-100.
Quality Control
Exonuclease I is tested in degradation of single-stranded DNA and is free of detectable RNase, endonuclease, and double-stranded exonuclease activities.
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