| Ribonuclease H specifically degrades only the RNA strand in RNA-DNA hybrids. It does not hydrolyze the phosphodiester bonds within single-stranded or double-stranded DNA and RNA. Unit Definition: One unit catalyzes the formation of 1nmol of acid soluble products in 20min at 37C. Enzyme activity is assayed in the following mixture: 20mM Tris-HCl (H 7.8), 40 mM KCl, 8mM MgCl2, 1mM DTT, 24uM [3H]-POLY(A)-POLY(dT), 0.03mg/ml BSA AND 4% Glycerol. 10X Reaction buffer (Included): R2021A Supplied as a liquid in 200mM Tris HCl, pH 8.0, 400mM KCl, 80mM MgCl2, 10mM DTT. Applications: Suitable for use in Removal of mRNA prior to the synthesis of the 2nd strand of cDNA, Removal of mRNA poly(A) sequences after hybridization with oligo(dT), Site-specific cleavage of RNA and Studies of in vitro polyadenylation rection products. Inhibition and Inactivation: -Inhibitors: metal chelators, SH-blocking reagents. -Inactivated by heating at 65C for 10min. Endodeoxyribonuclease Assay: No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 10 units of Ribonuclease H with 1ug of pBR322 DNA in 50ul of reaction buffer for 1 hour at 37C. Exodeoxyribonuclease Assay: 0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 10 units of Ribonuclease H with 1ug of sonicated E.coli [3H]-DNA in 50ul of reaction buffer for 1 hour at 37C. Ribonuclease Assay: 0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 10 units of Ribonuclease H with 1ug of [3H]-RNA in 50ul of reaction buffer for 1 hour at 37C. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
