FAQ: Can DNA Polymerase I (E. coli) 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), T4 DNA Polymerase (NEB# M0203) or Mung Bean Nuclease (NEB# M0250) are the best choices to remove 3' overhangs.