FAQ: Can the KLD reaction be incubated longer or at higher temperature?

The KLD reaction can be incubated for up to 60 minutes at room temperature. Alternatively, to increase efficiency of the DpnI digestion, the KLD reaction can be incubated at room temperature for 5 minutes, followed by 30-60 minutes at 37C. The room temperature incubation is essential for the activity of the kinase and ligase enzymes.