Jun 26, 2023
Version 2
NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546) V.2
- 1New England Bioloabs
External link: https://www.neb.com/products/e7595-nebnext-ultra-ii-ligation-module#Protocols,%20Manuals%20&%20Usage_Manuals
Protocol Citation: Juliet Bonnevie 2023. NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxmbnkl8j/v2Version created by Juliet Bonnevie
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 16, 2023
Last Modified: June 26, 2023
Protocol Integer ID: 83541
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.
Guidelines
Safe Stop Point: This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
Caution: Signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.
Color: A color listed before or after a reagent name indicates the cap color of the reagent to be added.
Adaptor Dilution Guidelines
The appropriate adaptor dilution for your sample input and type may need to be optimized experimentally. The dilutions provided here are a general starting point.
Table 1.1: Adaptor Dilution
| A | B | C | |
| Input | Adaptor Dilution (Volume of adaptor: Total volume) | Working Adaptor Concentration | |
| 1 µg–101 ng | No Dilution | 15 μM | |
| 100 ng-5 ng | 10-Fold (1:10) | 1.5 μM | |
| less than 5 ng | 25-Fold (1:25) | 0.6 μM |
Materials
MATERIALS
Ligation EnhancerNew England BiolabsCatalog #E7374 in Kits E7370 or E7445
NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
STEP MATERIALS
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Adaptor for IlluminaNew England Biolabs
Materials that may be needed that are not included in this kit:
Tris-HCL Buffer
10 mM NaCl
Protocol materials
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
In Materials, Materials, Step 4
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
Materials, Step 1
NEBNext Adaptor for IlluminaNew England Biolabs
Materials, Step 1
Ligation EnhancerNew England BiolabsCatalog #E7374 in Kits E7370 or E7445
Materials
NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
Materials
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
In Materials, Materials, Step 1
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.
Ligation/End Prep
Ligation/End Prep
30m
30m
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.
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Adaptor for IlluminaNew England Biolabs
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 00:15:00 in a thermal cycler with the heated lid off.
15m
Add 3 µl of (red) USER® Enzyme to the ligation mixture from Step 3.
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
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 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.
Mix well and incubate at 37 °C for 00:15:00 with the heated lid set to ≥ 47 °C .
15m
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.