Jun 16, 2023
Version 2
NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II FS DNA Module (NEB # E7810) V.2
- 1New England Biolabs
External link: https://www.neb.com/products/e7595-nebnext-ultra-ii-ligation-module#Protocols,%20Manuals%20&%20Usage
Protocol Citation: New England Biolabs, Juliet Bonnevie 2023. NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II FS DNA Module (NEB # E7810). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld3xw9g5b/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 08, 2023
Last Modified: June 22, 2023
Protocol Integer ID: 83069
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 point.
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 2.1: Adaptor Dilution
| A | B | C | |
| Input | Adaptor Dilution (Volume of adaptor: Total volume) | Working Adaptor Concentration | |
| 100 ng–500 ng | No Dilution | 15 μM | |
| 5 ng–99 ng | 10-Fold (1:10) | 1.5 μM | |
| less than 5 ng | 25-Fold (1:25) | 0.6 μM |
Materials
MATERIALS
NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
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
Ligation EnhancerNew England BiolabsCatalog #E7374 in Kits E7370 or E7445
NEBNext Adaptor for IlluminaNew England Biolabs
Materials that you may need that are not provided with this kit include:
Tris-HCL Buffer (pH 7.5)
10 mM NaCl
Protocol materials
Ligation EnhancerNew England BiolabsCatalog #E7374 in Kits E7370 or E7445
Materials, Step 1
NEBNext Adaptor for IlluminaNew England Biolabs
Materials, Step 1
NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
Materials
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
Materials
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
Materials
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
In Materials, Materials, Step 4
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
Materials, Step 1
Before start
Starting Material: 100 pg–500 ng fragmented, end repaired and dA-Tailed DNA generated using the NEBNext Ultra II FS DNA Module (NEB #E7810)
Note
Caution: If DNA input is < 100 ng, dilute the (red) NEBNext Adaptor for Illumina in 10 mM Tris-HCl, pH 7.5-8.0 with 10 mM NaCl as indicated in Table 2.1.
DNA Ligation/End Prep
DNA Ligation/End Prep
30m
30m
Add the following components directly to the FS Reaction Mixture:
| A | B | |
| Component | Volume | |
| FS Reaction Mixture | 35 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 | 68.5 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 Oligo kit options, which can be found at www.neb.com/oligos
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
Ligation EnhancerNew England BiolabsCatalog #E7374 in Kits E7370 or E7445
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. We do not recommend premixing 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 50 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 thermocycler with the heated lid off.
15m
Add 3 µl of (red) USER Enzyme to the ligation mixture.
USER Enzyme (Multiplex Oligos for Illumina)New England BiolabsCatalog #E7338
Note
Note: Steps 4 and 5 are only required for use with 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 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 #E7805/#E6177 manual for recommended size selection/cleanup and PCR amplification protocols.
Note
Safe Stop Point: Samples can be stored overnight at –20°C.