G 25 sephadex column

Radiolabeled probes for EMSAs were generated as described before (43 (link)). Uniformly labeled 126-nt blunt dsRNA was generated by in vitro transcription of T7 promoter-flanked PCR fragments using T7 RNA polymerase in the presence of α-³²P-[UTP]. After annealing of the two radiolabeled RNA strands, unincorporated nucleotides were removed using a G-25 Sephadex column (Roche) and dsRNA was purified from an 8% native polyacrylamide gel. Synthetic 21-nt siRNAs containing 2-nt 3′ overhangs and 19-nt blunt dsRNAs (43 (link)) were end-labeled with γ-³²P-[ATP] using T4 polynucleotide kinase (Roche) and purified on a G-25 Sephadex column.
EMSAs were performed as described previously (11 (link)). Briefly, radiolabeled 126-nt long dsRNA (5 ng), 19-nt dsRNA or siRNA duplexes (1 nM) were incubated with different concentrations of recombinant proteins for 1 h at room temperature. Long dsRNA and siRNA EMSAs were analyzed on 6% and 12% native polyacrylamide gels, respectively. Gels were exposed to a Kodak Biomax XAR film and radioactive signals were quantified with ImageJ software.

Detailed design information and probe sequences are listed in the (Additional file 1: Table S1). ssDNAs were purchased from Integrated DNA Technologies (Coralville, IA). Probes were generated by hybridization of equimolar amounts of complementary ssDNAs in 1× STE buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA), heating to 95 °C for 5 min, followed by incubation at room temperature for 1 h. After hybridization, dsDNA probes were 5′-radiolabeled with 10 pmoles of [γ-³³P] ATP using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). Radiolabeled DNA probes were purified from unincorporated label with G-25 Sephadex columns (Roche Applied Science; Indianapolis, IN).