Figure 2.

Multiple annealing conditions resulting in similar EMSA distributions. In the presence of monovalents alone (50 mM NaCl) or in the presence of divalents (12 mM MgCl₂) ³²P-labeled ribozyme (fully synthetic 2′-O-methyl construct used for mass spectrometry analysis) was subjected to the following annealing protocols: (1) 25°C for 20 min; (2) 70°C, 2 min, 25°C, 20 min; (3) 90°C, 2 min, 25°C 20 min; (4) 90°C, 2 min, 70°C, 2 min, 65°C, 2 min, 25°C, 20 min; (5) 90°C, 2 min, 25°C, 10 min repeated three times, then 70°C, 2 min, 25°C, 10 min repeated two times; (6) 90°C, 2 min, 55°C, 30 min, 25°C, 20 min. The distribution between T and B species was then analyzed by EMSA.