Applications:

Removal of 3'overhangs to form blunt ends

Fill-in of 5'overhangs to form blunt ends

T4 DNA polymerase catalyzes the synthesis of DNA in the 5' -> 3' direction and requires the presence of template and primer (i.e. the overhang). This enzyme has also a 3' -> 5' exonuclease activity.

Reaction buffer:

T4 DNA Polymerase is active in all four NEB-buffers when supplemented with dNTPs.

1x T4 DNA Polymerase buffer: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9.

Blunt ending 3' and 5' overhangs

DNA x µl

T4 Buffer 5 µl (if DNA is already in an NEB buffer just supplement with that buffer)

dNTPs 4 µl of 5 mM Mix (100 µM each final)

T4 Polymerase 3 µl (1-3 U/ µg: avoid dealing with huge amounts of DNA)

H2O x µl

Total 50 µl

Incubate 20 min at 12°C