FIGURE 5.

Effects of temperature, salt, and pH on single-vessel recombination/ligation. (A) The gel shows the higher molecular-weight products from recombination/ligation of B16–19RC. Reactions performed in the pH buffers indicated and in 10 mM MgCl₂/5 mM CaCl₂ (except lane 2) for the times and temperatures indicated. Lane 1 is the equivalent reaction as lane 10 in Figure 4. (B) The empirical response of pH to temperature of the buffers (Tris, nominal pH = 8.0; EPPS, nominal pH = 7.6) under simulated reaction conditions of cooling from 70°C to 20°C. The recombination and ligation temperatures employed in the gel experiment (60°C or 50°C and 22°C, respectively) are indicated by thick dashed lines.