5 32p end labeled 18 mer dna primer

Cellular dNTPs were measured by HIV-1 RT-based dNTP assay as described (54 (link)). Briefly, 2 × 10⁶ cells for each cell type were washed with PBS and resuspended in ice-cold 65% methanol. To lyse, cells were vortexed for 2 min and incubated at 95 °C for 3 min. Cells were centrifuged at 14,000 rpm for 3 min, and the supernatant was dried using a speed vac. The dried sample was resuspended in water and diluted to be within the linear range of the assay. 5′ 32P-end-labeled 18-mer DNA primer (5′-GTCCCTCTTCGGGCGCCA-3′; Integrated DNA Technologies) was individually annealed to one of four 19-mer DNA templates (3′-CAGGGAGAAGCCCGCGGTN-5′; Integrated DNA Technologies), where N represents the nucleotide variation at the 5′ end. Reactions (20 μl) contained 200 fmol template/primer, 4 μl of purified RT (HIV-1 HXB2), 25 mM Tris–HCl, pH 8.0, 2 mM dithiothreitol, 100 mM KCl, 5 mM MgCl₂, and 10 μM oligo (dT), and cellular dNTP extracts. Water or 0.5 mM dNTP mix was used as a negative and positive control, respectively. Reactions were incubated at 37 °C for 5 min and stopped by adding 10 μl 40 mM EDTA and 99% (v/v) formamide followed by incubation at 95 °C for 2 min. Reactions were resolved on a 14% urea-PAGE gel (AmericanBio, Inc) and analyzed using PharosFX molecular imager. Image Lab software, version 5.1.2, (Bio-Rad) was used to quantify single-nucleotide extensions products.