Protocol for use with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.
Colored bullets indicate the cap color of the reagent to be added to a
Stopping points in the protocol.

Starting Material:
500 pg–1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, 10 mM Tris-HCl, pH 8.0 or 0.1X TE can be used.

1.1 NEBNext End Prep
  1. Mix the following components in a sterile nuclease-free tube:
     (green) NEBNext Ultra II End Prep Enzyme Mix 3 μl
     (green) NEBNext Ultra II End Prep Reaction Buffer 7μl
    Fragmented DNA 50 μl
    Total volume 60 μl

  2. Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix throughly. Perform a quick spin to collect all liquid from the sides of the tube.

    Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.

  3. Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
    30 minutes @ 20°C
    30 minutes @ 65°C
    Hold at 4°C

    If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.

  4. Proceed directly to NEBNext Ultra II Ligation Module NEB #E7595.