NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546)

Juliet Bonnevie

Published: 2023-06-26 DOI: 10.17504/protocols.io.kxygxmbnkl8j/v2

Abstract

This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext^®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina^®-compatible library construction.

The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

Before start

Starting Material:

500 pg–1 µg fragmented DNA that has been end repaired and dA-Tailed using the NEBNext End Repair/dA-Tailing Module (NEB #E7546).

Note

Caution: If DNA input is ≤ 100 ng, dilute the NEBNext Adaptor for Illumina in 10 mM Tris-HCl, pH 7.5-8.0 with with 10 mM NaCl as indicated in Table 1.1.

Steps

Ligation/End Prep

Add the following products directly to the End Prep Reaction Mixture:

A	B
Component	Volume (μl) Per Reaction
End Prep Reaction Mixture	60 µl
(red) NEBNext Adaptor for Illumina**	2.5 µl
(red) NEBNext Ultra II Ligation Master Mix*	30 µl
(red) NEBNext Ligation Enhancer	1 µl
Total Volume	93.5 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 the NEBNext Oligo kit options, which can be found at www.neb.com/oligos.

Note

Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. Do not premix the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.

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.

Note

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.

Incubate at 20°C for 0h 15m 0s in a thermal cycler with the heated lid off .

Add 3 µl of (red) USER® Enzyme to the ligation mixture from Step 3.

Note

Note: Steps 4 and 5 are only required for use with non-indexed NEBNext Adaptors. USER enzyme can be found in most NEBNext oligo kits, all options can be found on the Note: Steps 4 and 5 are only required for use with non-indexed NEBNext Adaptors. USER enzyme can be found in most NEBNext oligo kits, all options can be found on the www.neb.com/oligos page. If you are using the indexed UMI adaptor, USER is not needed. Please see corresponding manual for use with UMI on the E7395 product page under the protocols,manuals, and usage tab. page. If you are using the indexed UMI adaptor, USER is not needed. Please see corresponding manual for use with UMI on the E7395 product page under the protocols,manuals, and usage tab.

Mix well and incubate at 37°C for 0h 15m 0s with the heated lid set to ≥ 47°C.

DNA is now ready for size selection or cleanup.

Note

Note: Please see NEB #E7645/#E7103 manual for recommended size selection/cleanup and PCR amplification protocols.

Note

Safe Stop Point: Samples can be stored overnight at –20°C.