Ribonuclease H (RNase H) is an endoribonuclease that specifically degrades the RNA strand of an RNA-DNA hybrid to produce 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA.

Applications
Removal of mRNA during second-strand cDNA synthesis. Removal of poly(A) sequences from mRNA in the presence of oligo(dT). Oligodeoxyribonucleotide-directed cleavage of RNA.

Source
Purified from E. coli expressing the E. coli RNase H gene on a plasmid.

Performance and quality testing
Ribonuclease, nonspecific endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease.

Unit definition
One unit hydrolyzes 1 nmol of RNA in ³H-labeled poly(A)poly(dT) to acid-soluble material in 20 min at 37°C.

Unit reaction conditions
20 mM Tris-HCl (pH 7.5), 0.1 M KCl, 10 mM MgCl₂ , 0.1 mM DTT, 5% (w/v) sucrose, 0.5 nmol ³H-labeled poly(A)poly(dT), and enzyme in 50 μL for 20 min at 37°C.