Aug 14, 2023
NEBNext® Ultra™ II DNA PCR-free Libray Prep Kit for Illumina® (NEB #E7410S/L, #E7415S/L)
- 1New England Biolabs
External link: https://www.neb.com/products/e7410-nebnext-ultra-ii-dna-pcr-free-library-prep-kit-for-illumina#Protocols,%20Manuals%20&%20Usage_Manuals
Protocol Citation: New England Biolabs 2023. NEBNext® Ultra™ II DNA PCR-free Libray Prep Kit for Illumina® (NEB #E7410S/L, #E7415S/L). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkoebwv5r/v1
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 27, 2023
Last Modified: August 14, 2023
Protocol Integer ID: 84101
Abstract
The NEBNext Ultra II DNA PCR-free Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform without PCR amplification. The fast, user-friendly workflow also has minimal hands-on time.
Each kit component must pass rigorous quality control strandards, and for each new lot the entire set of reagents is funtionally validated together by construction and sequencing of indexed libraries on Illumina sequencing platform.
For larger volume requiremnts, customized and bulk packaging is abailable by pruchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information.
Materials
The Library Kit Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7410S/#E7415S) and 96 reactions (NEB #E7410L/#E7415aL). All reagents should be stored at -20°C. Colors in parenthesis represent the color of the cap of the tube containing the reagent.
Package 1: Store at -20°C
(green) NEBNext Ultra II End Prep Enzyme Mix
(green) NEBNext Ultra II End Prep Reaction Buffer
(red) NEBNext Ultra II Ligation Master Mix
(red) NEBNext Ligation Enhancer
Package 2: Store at room temperature. Do not freeze.
Supplied only with NEBNext Ultra II DNA PCR-free Library Prep with Sample Purification Beads, NEB #E7415.
NEBNext Sample Purification Beads.
Required Materials Not Included
- 80% Ethanol (freshly prepared)
- Nuclease-free Water
- 0.2 ml thin wall PCR tubes
- DNA LoBind® Tubes (Eppendorf #022431021)
- NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) NEB #E7395
- Magnetic rack (NEB #S1515), magnetic plate (Alpaqua® cat. #A001322) or equivalent
- PCR machine
- Vortex
- Microcentrifuge
- Bioanalyzer®, TapeStation® (Agilent Technologies, Inc.) or similar instrument and consumables.
For NEB #E7410 only:
- SPRIselect Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
Before start
Starting Material: 250 ng-1 μg purified, genomic DNA sheared to 400 bp range. We recomment 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, sample can be diluted with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
NEBNext End Prep
NEBNext End Prep
1h
1h
Ensure that the Ultra II End Prep Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
Add the following components to a 0.2 ml thin wall PCR tube on ice:
| A | B | |
| COMPONENT | VOLUME PER ONE LIBRARY | |
| Fragmented DNA | 50 μl | |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 7 μl | |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 3 μl | |
| Total Volume | 60 μl |
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 for 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.
Place in a thermal cycler, with the heated lid set to 75°C, and run the following program:
00:30:00 at 20 °C
00:30:00 at 65 °C
Hold at 4 °C
Proceed immediately to the next section once the reaction temperature reaches 4°C.
1h
Adaptor Ligation
Adaptor Ligation
15m
15m
Add the following components directly to the End Prep Reaction Mixture:
| A | B | |
| COMPONENT | VOLUME | |
| End Prep Reaction Mixture (Step 4) | 60 μl | |
| NEBNext UMI Adaptors 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 |
* The NEBNext UMI adaptors are provided in NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1, NEB #E7395). Please refer to the NEB #E7395 manual for valid barcode combinations.
** Mix the Ultra II Ligation Master Mix by pipetting up and down several times times prior to adding to the reaction.
Note
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours at 4°C. Do not premix the adaptor with the Ligation Master Mix and Ligation Enhancer.
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 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. Move immediately to the next step or place your sample at -20 °C .
Safe Stopping Point: Samples can be stored overnight at -20 °C .
15m
Size Selection of Adaptor-ligated DNA
Size Selection of Adaptor-ligated DNA
1h 15m
1h 15m
Note
The volumes of NEBNext Sample Purification or SPRIselect Beads provided here are for use with the sample contained in the exact buffer at this step (93.5 μl; Step 5). These volumes may not work properly for a size selection at a different step in the workflow, or if this is a second size selection. For size selection of samples contained in different buffer conditions the volumes may need to be experimentally determined.
Caution: The following cleanup protocol is for libraries with ~350 bp or ~450 bp inserts only (Step 20). Size selection conditions were optimized with NEBNext Sample Purification Beads and SPRIselect beads. However, AMPure XP beads can be used following the same conditions. If using AMPure XP beads, please allow the beads to warm to room temperature for at least 00:30:00 before use.
30m
Bring the volume of the reaction to ~100 µL by adding 7 µL 0.1X TE (dilute 1X TE Buffer 1:10 with water).
Vortex NEBNext Sample Purification Beads or SPRIselect Beads to resuspend.
Add 50 µL of resuspended NEBNext Sample Purification Beads or SPRIselect beads to the 100 μl sample from Step 9. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
Incubate samples for at least 00:05:00 at room temperature.
5m
Place the tube/ plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 00:05:00 (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets.
Caution: do not discard beads.
5m
Add 200 µL of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
Repeat the previous step once. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
Air dry the beads for up to 00:05:00 while the tube/ plate is on the magnetic stand with the lid open.
Caution: Do not overdry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
5m
Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads into 102 µL of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for 00:02:00 at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
2m
Place the tube/ plate on a magnetic stand. After 00:05:00 (or when the solution is clear), transfer 100 µL to a new tube.
5m
Add appropriate amounts of resuspended Sample Purification or SPRIselect Beads to the sample for the desired insert sizes. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
| A | B | |
| INSERT SIZE | BEADS VOLUME | |
| 350 bp | 65 μl | |
| 450 bp | 58 μl |
Incubate samples for at least 00:05:00 at room temperature.
5m
Place the tube/ plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 00:05:00 (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets.
Caution: do not discard beads.
5m
Add 200 µL of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
Repeat the previous step once. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
Air dry the beads for up to 00:05:00 while the tube/ plate is on the magnetic stand with the lid open.
Caution: Do not overdry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
5m
Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads into 22 µL of 0.1X TE. Mix well on a vortex mixer by pipetting up and down 10 times. Incubate for 00:02:00 at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
2m
Place the tube/ plate on a magnetic stand. After 00:05:00 (or when the solution is clear), transfer 20 µL to a new tube.
5m
Quantitate the library using qPCR (NEBNext Library Quant Kit for Illumina, NEB #E7630S/L).
Verify fragment size by checking the library size distribution on an Agilent Bioanalyzer or TapeStation. Run 1 µL library on a DNA High Sensitivity Chip (Bioanalyzer) or High Sensitivity D5000 ScreenTape® (TapeStation). See Figure 1 for an example.
Safe Stopping Point: It is safe to store the library at -20 °C .
Figure 1: Examples of Ultra II DNA PCR-free libraries on a Bioanalyzer. The PCR-free libraries migrate slower due to the single strand regions of the adaptors, thus appearing significantly larger than the actual fragment sizes.