Deoxyribonuclease I (DNase I) is an endonuclease isolated from bovine pancreas that digests double- and single-stranded DNA into oligo- and mononucleotides. Using the Reaction Buffer provided, DNA is removed from RNA preparations in a 15 minute digestion at room temperature. The DNase I is then inactivated by heating with the Stop Solution. Heating also denatures hairpins in the RNA, so the RNA can be used directly in reverse transcription.

Many commercial DNase I formulations are contaminated with residual RNases. This RNase contamination can destroy or degrade valuable RNA samples prior to reverse transcription. Laboratory comparisons have shown that Sigma′s Amplification Grade DNase I demonstrates lower RNase activity than that from several leading molecular biology product suppliers.