FAQ: Can phi29 DNA Polymerase be used to fill in 3' overhangs?

No, DNA polymerases cannot fill in 3' overhangs. To create blunt ends 3' overhangs must be removed. DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) or T4 DNA Polymerase (NEB# M0203) are the best choices to remove 3' overhangs. Mung Bean Nuclease (NEB# M0250) can also be used to remove 3´ overhangs.