FAQ: Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?

DNA Polymerase I, Large (Klenow) Fragment 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.