Enzymes for Molecular Biology: Enzymes for Molecular Biology

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.

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 ExonucleaseI. 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.

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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Protocols for: Exonuclease I, E. coli

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Lucigen"s product page, where the latest protocol is available.

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Exonuclease I Protocol

(catalogue number X40501K / X40505K / X40520K)

Please note: all protocols off site are the responsibility of the products supplier

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References

1. Lehman, I.R. and Nussbaum, A.L. (1964) J. Biol. Chem. 239, 2628.
2. Kushner, S.R. et al. (1971) Proc. Natl. Acad. Sci. USA 68, 824.
3. Kushner, S.R. et al. (1972) Proc. Natl. Acad. Sci. USA 69, 1366.

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Applications

• Removal of residual ssDNA, including oligos, from reaction mixes.
• Removal of ssDNA from nucleic acid mixtures

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