Ribonuclease H (RNase H) is an endoribonuclease which specifically degrades the RNA strand of an RNA/DNA hybrid, leaving the DNA strand and unhybridized RNA intact. E. coli RNase H (RNaseHI) was over-expressed in E. coli as a recombinant protein and the protein then purified. MW is 17.6 kDa.
- Removal of mRNA in DNA/RNA hybrid prior to the synthesis of the second strand of cDNA (1, 2)
- Removal of poly (A) tails from mRNA after hybridization with oligo (dT) (3)
- Oligodeoxyribonucleotide-directed site-specific cleavage of RNA (4)
Form: 50 units/ul in 20mM Tris-HCl (pH 7.5), 100mM KCl, 1mM DTT, 50% Glycerol
Specific Activity: 100,000 units/mg protein
Unit Definition: 1 unit is defined as the amount of the enzyme that hydrolyzes 1 nmol of the RNA in 3H labeled M13 DNA/RNA hybrid to acid-soluble ribonucleotides in 20 min at 37°C.
Quality: Greater than 95% protein determined by SDS-PAGE (Fig. 1, CBB staining). Endo- and exo-DNase activities and RNase activity were not detected with 100 U/ml RNaseH in 50 ul reaction at 37°C.
Reagents Supplied with Enzyme: 10X RNaseH Reaction Buffer: 100 mM Tris-HCl (pH 8.0), 100 mM MgCl2, 500 mM NaCl, 10 mM DTT, 500 ug/ml BSA (Bovine Serum Albumin)
Caution: To avoid contamination of trace amounts of nucleic acids in BSA, use reaction buffer that
does not contain BSA and use RNaseH at higher concentrations.
Data Link: UniProtKB/Swiss-Prot P0A7Y4 (RNH_ECOLI)
- Gubler U (1987) “Second-strand cDNA synthesis: mRNA fragments as
primers.” Method Enzymol 152: 330-335 PMID: 3309563
- Sambrook J & Russell DW (2001) Molecular Cloning, Chapter 11
“Preparation of cDNA Libraries and Gene Identification”. CSHL Press
- Vournakis JN et al (1975) “Electrophoretic patterns of deadenylylated
chorion and globin mRNAs.” Proc Natl Acad Sci USA 72: 2959-2963
- Donis-Keller H (1979) “Site specific enzymatic cleavage of RNA.”
Nucleic Acids Res. 7: 179-192 PMID: 386279
Fig.1 SDS-PAGE of E. coli RNaseH