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. 2014 May 3;42(10):6774–6785. doi: 10.1093/nar/gku307

Figure 3.

Figure 3.

Component optimization for improved iSAT activity from p16S-HH and p23S-HH constructs. (A) Ratio optimization of co-transcribed plasmids included in p16S-HH/p23S-HH iSAT reactions for luciferase production. (B) Ratio optimization of T7 RNAP and plasmid concentrations included in p16S-HH/p23S-HH iSAT reactions for luciferase production. T7 RNAP was diluted with storage buffer to maintain equivalent salt and buffer concentrations in all reactions. A mix of plasmids was similarly diluted with water. Plasmid concentrations shown are for p23S-HH, with a constant ratio of 2:1:10 for pK7Luc:p16S-HH:p23S-HH, as determined from (A). For panels (A) and (B), values show average luciferase concentrations above background for two independent reactions. (C) Effect of TP70 concentration on iSAT activity using p16S-HH and p23S-HH with optimized plasmid and T7 RNAP concentrations. TP70 was diluted with storage buffer to maintain equivalent salt and buffer concentrations in all reactions. (D) Summary of improvements in iSAT activity due to plasmid and polymerase concentration optimization. For panels (C) and (D), values show average luciferase concentrations above background with error bars representing s.d. for at least three independent reactions. (E) RNA denaturing gel of iSAT reactions utilizing p16S-HH and p23S-HH plasmids and optimized reaction conditions.