Ribonuclease H (RNase H)

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.\n\nUnit Definition:\nOne 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.\n\n10X Reaction buffer (included):\nR2021A\nSupplied as a liquid in 200mM Tris HCl, pH 8.0, 400mM KCl, 80mM MgCl2, 10mM DTT.\n\nApplications:\nSuitable 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.\n\nInhibition and Inactivation:\nInhibitors: metal chelators, SH-blocking reagents.\n-Inactivated by heating at 65C for 10min.\n\nEndodeoxyribonuclease Assay:\nNo 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 \nin 50ul of reaction buffer for 1 hour at 37 degrees C. \n\nExodeoxyribonuclease Assay:\n0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 10 units of Ribonuclease H with 1ug of sonicated \nE.coli [3H]-DNA in 50ul of reaction buffer for 1 hour at 37 degrees C. \n\nRibonuclease Assay:\n0% 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 37 degrees C.\n\nStorage and Stability:\nFor long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. 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.

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