Optimal reaction conditions depend on the downstream step. For library construction, low temperatures such as 16 degrees Celsius and long incubation times, 12-16 hours, provide the best reaction yields. This allows you to maximize library complexity. Complexity is not important for subcloning of a single insert into a vector; therefore, you can accomplish this with a much shorter reaction time, 15 minutes or less, over a much broader range of temperatures, 16 degrees Celsius to 37 degrees Celsius.
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