Mulv rt

MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. HIV-1 NC was cloned into pET28b, between NdeI and XhoI, to give an N terminal His-tagged construct. This expression plasmid was transformed into BL21 cells which were grown in Luria-Bertani medium until OD₆₀₀ = 0.7, at which time expression was induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were supplemented with 50 µM Zinc acetate. Cells were grown overnight (20 h) at 16°C, collected by centrifugation and stored at −20°C until further use. Frozen cells were resuspended in purification buffer (500 mM NaCl, 50 mM Tris 8.3, 5 mM imidazole and 5 mM beta-mercaptoethanol). Cells were disrupted, centrifuged at 14 000 rpm and His-tagged protein was captured from the supernatant using a Nickel bead affinity purification column. Bound protein was washed in purification buffer supplemented with 30 mM imidazole and eluted in purification buffer supplemented with 300 mM imidazole. The sample was washed in a 3 kDa centricon in dialysis buffer (200 mM NaCl and 20 mM Tris 8.3) before further purification on a S200 size exclusion column equilibrated and run in dialysis buffer. Elutions containing NC were stored at −80°C.