TITLE: NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546)
AUTHORS: New England  Biolabs, Menna Teffera

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext</span><span style = "vertical-align:super;">®</span><span>Ultra II End Repair/dA-Tailing Module (NEB #</span><a href="https://www.neb.com/products/e7546-nebnext-ultra-ii-end-repair-da-tailing-module" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">E7546</span></a><span>), for Illumina</span><span style = "vertical-align:super;">®</span><span>-compatible library construction.</span></div><div class = "text-block"><span>The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #</span><a href="https://www.neb.com/products/e7546-nebnext-ultra-ii-end-repair-da-tailing-module" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">E7546</span></a><span>) or the NEBNext Ultra II FS DNA Module (NEB #</span><a href="https://www.neb.com/products/e7810-nebnext-ultra-ii-fs-dna-module" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">E7810</span></a><span>).</span></div></div>

[STEPS]
?. [Ligation/End Prep]
Add the following products directly to the End Prep Reaction Mixture:  AB1ComponentVolume2End Prep Reaction Mixture60 µl3(red) NEBNext Ultra II Ligation Master Mix*30 µl4(red) NEBNext Ligation Enhancer1 µl5(red) NEBNext Adaptor for Illumina**2.5 µl6Total Volume93.5 µl* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.** The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535, and #E6609) Oligos for Illumina.
AB1ComponentVolume2End Prep Reaction Mixture60 µl3(red) NEBNext Ultra II Ligation Master Mix*30 µl4(red) NEBNext Ligation Enhancer1 µl5(red) NEBNext Adaptor for Illumina**2.5 µl6Total Volume93.5 µl
Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.
?. [Ligation/End Prep]
Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.
?. [Ligation/End Prep]
Incubate at  for  in a thermocycler with the heated lid off.
20 °C
?. [Ligation/End Prep]
Add 3 µl of (red) USER™ Enzyme to the ligation mixture from Step 3.
Note: Steps 4 and 5 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600 and #E6609) Oligos for Illumina.
?. [Ligation/End Prep]
Mix well and incubate at  for  with the heated lid set to ≥ .
37 °C
47 °C
?. [Ligation/End Prep]
DNA is now ready for size selection or cleanup.
Note: Please see NEB #E7645 manual for recommended size selection/cleanup and PCR amplification protocols.
Safe Stop Point: Samples can be stored overnight at –20°C.