Hiload superdex s200

The D4A RNA construct was transcribed and purified as described above, but at a larger scale. The gel bands corresponding to the transcribed D4A were visualized by UV-shadowing, excised from the gel, and the D4A RNA was electro-eluted overnight at 4 °C using an EluTrap system (Whatman). The RNA was ethanol precipitated, washed with 70% ethanol, and the resulting RNA pellet was dissolved in 500 μL of a buffer containing 10 mM MES pH 6.0, 200 mM KCl and 1 mM EDTA. Before complex assembly, D4A was heated to 95 °C for 2 min and then snapped cooled on ice. D4A was then mixed with E.r. RT in buffer H (25 mM K-HEPES pH 7.5, 2 M KCl, 10% glycerol and 1 mM DTT) at an equal molar ratio and the mixture was dialyzed against buffer I (25 mM K-HEPES pH 7.5, 200 mM KCl and 1 mM DTT) at 4 °C overnight. The complex was injected onto a HiLoad Superdex S200 gel-filtration column (GE Healthcare) equilibrated with buffer I and the peak fractions were pooled, concentrated, flash-frozen by N_2(l) and stored at −80 °C.