Protocol for blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

  1. DNA should be dissolved in 1X reaction buffer* supplemented with 100 μM of each dNTP.
  2. Add 1 unit T4 DNA Polymerase per microgram DNA.
  3. Incubate 15 minutes at 12°C.
  4. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes (1,2).
CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´ → 5´ exonuclease activity of the enzyme.

* T4 DNA Polymerase can be used in NEBuffers r1.1, r2.1, and rCutSmart™ Buffer  as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer r2.1. Recombinant Albumin (rAlbumin) supplementation is recommended when using a buffer that does not already contain albumin. 

  1. Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. In F. M. Ausebel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl (Ed.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc.
  2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.