Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

RT reactions with retroviral reverse transcriptases involved a pre-annealing step of the RNA template (100 ng): DNA Primer (2 pmol) (Supplementary Table S1 ), in the presence of the dNTP substrate (with the specific concentrations of each indicated in the main text), with the presence or absence of antisense where indicated (2, 20 or 200 pmol), in 10 l reaction volumes. With wtSL or MUTsl RNA templates, incubation was at 65 • C for 5 min before chilling on ice. For G-rich RNA templates with potential to form structure formation larger than that of the model stem-loop wtSL, the annealing mix had in addition 10 mM KCl and the annealing step was at 95 • C for 30 s with a 1 • C temperature decrease (from 95 • C to 16 • C) every 30 s. On completion one of several different 10 l reaction mixes was added and incubated for 50 min at the temperature indicated. One reaction mix contained 100 units SuperScript™ Script™ III buffer and 20 mM DTT-incubation 37 • C. On completion a further incubation, which was at 85 • C, followed for 5 min.