Exonuclease III is a 3' to 5' exonuclease that removes mononucleotides from the 3´-hydroxyl termini of double-stranded DNA or DNA-RNA hybrids. Because a set number of mononucleotides are generated during each degradation, double-stranded DNA degradation is progressive. Exonuclease III will degrade double-stranded DNA containing a blunt end, a 5'-overhang or a nick, but it will not degrade DNA with a 3'-overhang containing 4 or more bases. This 3' to 5' exonuclease can be inactivated by heating it at 65°C for 5 minutes. Exonuclease III is supplied in a buffer of 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.5 mM DTT and 50% glycerol.