Amersham typhoon rgb scanner

10 pmol of either ssRNA I or ssRNA A was incubated in the presence or absence of both Mg²⁺ at a 10 mM final concentration and/or 9 pmol recombinant eEndoV (New England Biolabs) in a total volume of 10 μL. Final buffer conditions in all reactions were 10 mM Tris, 125 mM NaCl, 15 μM EDTA, 150 μM DTT, 0.025% Triton X-100, 30 μg/ml BSA, 7% glycerol, pH 7.4. Reactions were incubated for 1 hour at 25 °C, followed by a 10 min heat inactivation at 85 °C. Reaction products were separated using 10% denaturing PAGE, and gels were imaged with a GE Amersham Typhoon RGB scanner using 635 nm excitation laser and the Cy5 670BP30 emission filter. To test cleavage in the presence of Ca²⁺, 10 pmol of ssRNA I was incubated with 840 nM eEndoV with either 10 mM MgCl₂ or variable amounts of CaCl₂ (0, 0.1, 0.5, 1, 2.5, 5, 10 and 20 mM) in a total volume of 50 μL. Final buffer conditions in all reactions were 19 mM Tris, 137 mM NaCl, 3 mM KCl, 15 μM EDTA, 150 μM DTT, 0.025% Triton X-100, 30 μg/ml BSA, 7% glycerol, pH 7.4. Reactions were incubated at room temperature for 3 hours, after which a 3 μL sample was taken for 10 % denaturing PAGE analysis as described above.

To assess duplex formation, 100 pmol of each RNA pair (untreated or glyoxalated) were mixed together in 19 mM Tris, 137 mM NaCl, 3 mM KCl, pH 7.4. Mixtures were heated to 95 °C for 5 minutes and slowly cooled to room temperature over the course of approximately 1 hour. 10 pmol of annealed construct was then loaded onto a 10% native non-denaturing polyacrylamide gel and imaged with a GE Amersham Typhoon RGB scanner.