FAQ: What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?

DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) and T4 DNA Polymerase (NEB #M0203) are the best choices for this application. DNA Polymerase I, Large (Klenow) Fragment can be used at 25°C or room temperature, but T4 DNA Polymerase must be used at 12°C due to its robust exonuclease. Both work well in a wide variety of buffers. Vent DNA Polymerase (NEB #M0254) and Deep Vent DNA Polymerase (NEB #M0258) can also be used but ThermoPol buffer must be used, the reaction temperature is high, and the enzyme cannot be heat inactivated. Mung Bean Nuclease (NEB #M0250) will chew back 3' overhangs but the strong exonuclease activity combined with the lack of polymerase activity yield a lower percentage of blunt ends.