DNase I is an endodeoxyribonuclease that digests both single- and double-stranded DNA. It recognizes and cleaves phosphodiester bonds, producing monodeoxynucleotides or single-/double-stranded oligodeoxynucleotides with a 5′ phosphate group and a 3′ hydroxyl group. The activity of DNase I is calcium-dependent and can be activated by divalent metal ions such as Mn²⁺ and Zn²⁺. The presence of 5 mM Ca²⁺ helps protect the enzyme from hydrolysis. In the presence of Mg²⁺, DNase I randomly cleaves any site on either DNA strand. When Mn²⁺ is present, the enzyme can simultaneously recognize and cleave both DNA strands at nearly the same site, producing either blunt-ended fragments or sticky ends with 1-2 nucleotide overhangs.

One unit is defined as the amount of enzyme which will completely degrade 1 μg of pBR322 plasmid DNA in 10 minutes at 37°C.

1. Prepare the reaction in an RNase-free tube:
RNA: X μg
10× DNase I Buffer: 1 μL
DNase I, RNase-free (5 U/μL): 1 U per μg RNA
ddH2O: Up to 10 μL
2. Incubate at 37°C for 15 minutes
3. Add 0.5 M EDTA to a final concentration of 2.5-5 mM to stop the reaction, then heat at 65°C for 10 minutes to inactivate DNase I. The sample can be directly used in downstream reactions such as reverse transcription.
Notes: EDTA is user-provided. Use 1 U per μg RNA, or 1 U if RNA < 1 μg. Keep enzyme on ice during handling.

RNase: 5 U of DNase I incubated with 1.6 μg MS2 RNA for 4 hours at 37°C shows no degradation as determined by agarose gel electrophoresis.

Ship with ice packs. Store at -20°C. Avoid repeated freeze-thaw cycles.