Typhon fla9500

For assembly of scaffolds used for primer extension experiments, the DNA and RNA oligonucleotides (1 mL, 20 mM each) were mixed and incubated at 95 C for 3 min. The mixture was placed immediately on ice for 1 min. Afterwards, 3 mL of 23 HB buffer were added, and the mixture was incubated at room temperature for at least 10 min. Prior to scaffold assembly, the used RNA was radioactively labelled on the 5 0 -end with [g-32P]ATP (10 mCi/mL, Hartmann Analytic) and T4 PNK (NEB), following purification via PAGE. The annealed scaffold was diluted to 2 mM with transcription reaction buffer (TRB) (20 mM Hepes, pH 7.6, 60 mM (NH 4 ) 2 SO 4 , 10 mM MgSO4, 10% glycerol, 2 mM DTT). 1 mL of diluted scaffold was incubated with 1 mL of Pol III (4 mM, also diluted in TRB) for at least 10 min at RT. Primer extension was initiated via addition of 7 mL TRB, supplemented with NTPs (1 mM each, final concentration). The reaction was incubated at 28 C for 20 min and stopped by adding 3 mL of formamide and boiling for 4 min. The reaction was subjected to PAGE using a denaturing (8M Urea) 17% polyacrylamide TBE gel. Radioactive signal was captured with a phosphor-imaging screen (Fujifilm) and detected with a Typhon FLA9500 (GE Healthcare) instrument.