RNase I degrades single-stranded RNA to nucleoside 3´ monophosphates via 2´,3´ cyclic monophosphate intermediates by cleaving between all dinucleotide pairs,2,3 unlike RNase A, which cleaves only after cytosine and uridine. In addition, the enzyme is completely inactivated by heating at 70°C for 15 minutes, eliminating the requirement to remove the enzyme prior to many subsequent procedures.
Unit Definition: One unit of RNase I degrades 100 ng of E. coli ribosomal RNA per second into acid-soluble nucleotides at 37°C under standard assay conditions.
Dilution and Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1 mM EDTA.
Quality Control: RNase I is free of detectable exo- and endodeoxyribonuclease activities.
|Figure 1. Removal of RNA from plasmid DNA preparations. Plasmid DNA was prepared from 1.5 mL of an overnight culture, suspended in 50 µL TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA), and treated with RNase A, RNase I, or no enzyme. Five µL of each reaction were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide. Lane 1, no RNase; Lane 2, 1.5 U of RNase I; Lane 3, 20 µg/mL RNase A; MW, 1-kb ladder. Arrow denotes small undigested RNA.|