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| | RNase H (RH) Assay |
| Description: | HIV RT-associated RNase H activity was measured as previously described.[J. Med. Chem. 2014, 57:3223-3234] Briefly, the reaction mixture comprised Tris HCl (100 mL, 50 mm, pH 7.8) containingMgCl2 (6 mm), dithiothreitol (1 mm), KCl (80 mm), hybrid RNA/DNA 5'-GAUCU GAGCC UGGGA GCU-Fluorescin-3' (0.25 mm; HPLC, dry, QC: Mass Check; Metabion, Steinkirchen, Germany), 5'-Dabcyl-AGCTC CCAGG CTCAG ATC-3' (HPLC, dry, QC: Mass Check), and HIV-1 RT (20 ng, according to a linear range of dose-activity curve). The reaction mixture was incubated for 1 h at 37 °C, and the reaction was stopped by addition of EDTA; products were measured with a Victor 3 multilabel counter plate reader (model 1420-051; PerkinElmer) equipped with filters (lex=490 nm, lem=528 nm). |
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