NEBNext Ultra II DNA Library Prep Kit for Illumina
Instruction Manual
Before you get started: Input material must be between 500 pg – 1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. PLEASE ADJUST SAMPLE VOLUMES TO 50 μl! If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, dilute samples with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
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NEB #E7645S/L, #E7103S/L Version 6.1_5/20
Preparation of input material: Input material must be between 500 pg – 1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. PLEASE ADJUST SAMPLE VOLUMES TO 50 μl! If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, dilute samples with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
Done
Safe Stop! Click to receive information on how to store materials to pause
Note Click to receive additional information
Caution Click to receive additional information
Place reagents below on ice:
(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
(blue) NEBNext Ultra II Q5 Master Mix
Only for DNA input below 100 ng (place at room temperature to thaw):
NEBNext Adaptor Dilution Buffer (10 mM Tris-HCl, pH 7.5 – 8.0 with 10 mM NaCl)
Nuclease-free Water
0.1x TE
NEBNext Sample Purification Beads OR SPRI Select Reagent Kit OR AMPure XP Beads
80% EtOH (freshly prepared)
Preparation
(red) NEBNext Adaptor for Illumina
(red) USER Enzyme
Back
Place reagents below at room temperature:
NEBNext Multiplex Oligos
Prepare End Prep / dA-Tailing Master Mix
Choose Amounts
Choose the number of samples to be processed:
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93
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96
79
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Choose the desired reactions accounting for dead volumes:
NEBNext End Prep
1.1 Prepare End Prep /dA-Tailing Master Mix by adding the following components to a sterile nuclease-free tube
COMPONENT
VOLUME
3 μl
7 μl
count
amount1
amount2
1.2 Set a pipette to 50 μl and mix by pipetting 10 times, then quick spin
Mix by pipetting 10 times, then quick spin.
Module #E7546
End Repair/ dA-Tailing
Module #E7595
Adaptor Ligation
component of #E7103
Clean Up/ Size Selection
Module #M0544
PCR Enrichment
Clean Up
NEBNext Ultra II DNA Library Prep Kit for Illumina (#E7645)
End Repair / dA-Tailing
Set a pipette to:
((amount 1) +(amount2))*0.8
NOTE: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
Once thermal cycling is completed, quick spin samples to centrifuge condensate that has formed.
Add 10 μl of End Prep /dA-Tailing Master Mix to each sample (fragmented DNA):
Fragmented DNA
50 μl
End Prep / dA-Tailing Master Mix
10 μl
Total Volume
60 μl
X
Mix the NEBNext Ultra II End Prep Enzyme Mix by flicking 10 times.
Mix NEBNext Ultra II End Prep Enzyme Mix
Mix the NEBNext Ultra II End Prep Reaction Buffer by vortexing 10 seconds on full speed.
Mix NEBNext Ultra II End Prep Reaction Buffer
Quick spin both NEBNext Ultra II End Prep Reaction Buffer and NEBNext Ultra II End Prep Enzyme Mix.
Quick spin End Prep reagents
1.3 Place in a thermal cycler and run the following program
Lid set to ≥ 75°C
30 minutes @ 20°C
30 minutes @ 65°C
Hold at 4°C
Note: Once thawed, place Adaptor Dilution Buffer on ice.
SAFE STOP: If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
Quick spin after cycling
Adaptor Dilution / Ligation
2.2 Add the prepared Ligation Master Mix
End Prep Reaction Mixture (from step1)
2.5 μl
Prepared Ligation Master Mix
31 μl
93.5 μl
2.4 Place in a thermal cycler and run the following program
Prepare Ligation Mix by adding the following components to a sterile nuclease-free tube
30 μl
amount3
1 μl
amount4
((amount 3) +(amount4))*0.8
2.3 Set a pipette to 80 μl and mix by pipetting 15 times, then quick spin
Lid turned off
15 minutes @ 20°C
2.5 Addition of USER Enzyme
Add 3 μl of (red) USER Enzyme to the Ligation Reaction Mixture.
Ligation Reaction Mixture
USER Enzyme
96.5 μl
2.6 Set a pipette to 80 μl and mix by pipetting 10 times, then quick spin
Input
Less than 5 ng
100 ng – 5 ng
1 μg – 101 ng
Adaptor dilution (volume of adaptor: total volume)
Working adaptor concentration
No Dilution
15 μM
Quick spin both NEBNext Adaptor Dilution Buffer and NEBNext Adaptor.
Quick spin reagents for adaptor dilution
Mix the NEBNext Adaptor Dilution Buffer by vortexing 5 seconds on full speed.
Mix NEBNext Adaptor Dilution Buffer (Option 100 ng – 5 ng):
Option5b
Option5a
Make a 1:25 Dilution (Option < 5 ng):
Quick spin the NEBNext Adaptor.
Quick spin NEBNext Adaptor (Option < 5 ng):
Mix the NEBNext Adaptor Dilution Buffer by vortexing 5 seconds on full speed, followed by a quick spin.
Mix NEBNext Adaptor Dilution Buffer (Option < 5 ng):
Option100
Make a 1:10 Dilution (Option 100 ng – 5 ng):
2.1 Determine whether adaptor dilution is necessary by clicking on the available input amount below
NOTE: 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. Excess adaptor should be removed prior to PCR enrichment.
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 adding adaptor to a premix in the Adaptor Ligation Step.
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.
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.
NEBNext Adaptor Dilution Buffer
Set the pipette to:
amount pipette
Mix the diluted NEBNext Adaptor by pipetting 10 times, followed by a quick spin, and keep on ice until use.
amount
Mix the diluted NEBNext Adaptor by pipetting 10 times
Quick spin the NEBNext Ultra II Ligation Master Mix and the NEBNext Ligation Enhancer.
Quick spin reagents for Ligation
Mix the NEBNext Ultra II Ligation Master Mix by inverting 10 times, followed by flicking 10 times.
Mix NEBNext Ultra II Ligation Master Mix
2.2 Add the NEBNext Adaptor to the End Prep Reaction Mixture
62.5 μl
Mix the USER Enzyme by flicking 10 times, followed by a quick spin.
Mix USER Enzyme
Mix the NEBNext Ligation Enhancer by flicking 10 times.
Mix NEBNext Ligation Enhancer
2.2 While on ice, add the diluted NEBNext Adaptor to the End Prep Reaction Mixture
Diluted NEBNext Adaptor for Illumina
2.2 While on ice, add the prepared Ligation Master Mix
2.2 Add the diluted NEBNext Adaptor to the End Prep Reaction Mixture
SAFE STOP: Samples can be stored overnight at –20°C.
Mix the NEBNext Adaptor by flicking 10 times.
Mix NEBNext Adaptor (Option 100 ng – 5 ng):
Place in a thermal cycler and run the following program
Lid set to ≥ 47°C
15 minutes @ 37°C
Mix NEBNext Adaptor (Option < 5 ng):
Size Selection or Cleanup of Adaptor-ligated DNA
What is the input amount of your starting material?
Size Selection of Adaptor-ligated DNA
Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
Select your insert size!
Approximate insert size distribution
Approx. Final Library Size Distribution (insert + adaptor + primers)
Bead volume to be added (μl)
1st Bead Addition: 50
2nd Bead Addition: 25
1st Bead Addition: 40
2nd Bead Addition: 20
370 bp
1st Bead Addition: 30
2nd Bead Addition: 15
480 bp
1st Bead Addition: 25
2nd Bead Addition: 10
600 bp
1st Bead Addition: 20
750 bp – 800 bp
1st Bead Addition: 15
3A.5 Transfer supernatant
3A.3 Incubate
Incubate samples on bench top for at least 5 minutes at room temperature.
After 5 minutes, carefully transfer the supernatant to a new tube.
Mix beads with supernatant
3A.8 Discard supernatant
Transfer eluate
Transfer 15 μl to a new PCR tube for amplification.
3A.11 Dry the beads
Air dry the beads for 3 minutes and 30 seconds while the tube/plate is on the magnetic stand with the lid open.
3A.2 Add Beads to Ligation Reaction
Add the following volume of resuspended beads to each 96.5 μl Ligation Reaction:
bead1
Mix Beads with Ligation Reaction
3A.4 Separate beads from supernatant
Place the tube/plate on an appropriate magnetic stand and wait for 5 minutes to separate the beads from the supernatant.
3A.10 Second ethanol wash
3A.9 First ethanol wash
While in the magnetic stand, add 200 μl of 80% freshly prepared ethanol to the tube/plate, wait 30 seconds, then carefully remove and discard the supernatant with a p200 pipette.
Incubate
Incubate resuspended beads on bench top for at least 2 minutes at room temperature.
3A.12 Elution of DNA
Remove the tube/plate from the magnetic stand and elute the DNA from the beads by adding either 17 μl of 10 mM Tris-HCl or 17 μl of 0.1X TE.
Size Selection
500 bp – 700 bp
400 bp – 500 bp
300 bp – 400 bp
250 bp
200 bp
150 bp
> 50 ng
≤ 50 ng
3A.1 Vortex Beads
Mix by pipetting 10 times.
(96.5 + bead1)*0.8
Discard the beads.
3A.6 Add resuspend beads to supernatant
Add resuspended NEBNext Sample Purification Beads or SPRIselect Beads or AMPure XP Beads to each supernatant:
bead2
(96.5 + bead1 + bead2)*0.8
3A.7 Seperate beads from supernatant
Repeat prior step, i.e. while in the magnetic stand, add 200 μl of 80% freshly prepared ethanol to the tube/plate, wait 30 seconds, then carefully remove and discard the supernatant with a p200 pipette.
Remove residual ethanol traces
Mix
Mix by pipetting up and down 10 times.
3A.14 Separate beads from the eluate
Vortex NEBNext Sample Purification Beads or SPRIselect Beads or AMPure XP Beads for at least 1 minute until fully resuspended and no more clumps are visible.
3B.1 Vortex Beads
3B.2 Add Beads to Ligation Reaction
Add 87 μl of resuspended beads to the 96.5 μl of Ligation Reaction.
Set a pipette to: 150 μl
3B.3 Incubate
3B.4 Separate beads from supernatant
3B.5 Discard supernatant
After 5 minutes, carefully remove and discard the supernatant with a p200.
With a p20 remove residual supernatant from the bottom of the tube.
3B.6 First ethanol wash
3B.7 Second ethanol wash
Repeat prior step, i.e. while in the magnetic stand, add 200 μl of 80% freshly prepared ethanol to the tube/plate and wait for 30 seconds.
3B.8 Dry the beads
3B.9 Elution of DNA
3B.10 Mix
3B.11 Separate beads from the eluate
Quick spin, place the tube/plate on a magnetic stand and wait for 5 minutes to separate the beads from the eluate.
CAUTION If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use.
CAUTION Be careful to expel all of the liquid out of the tip during the last mix.
CAUTION Do not discard the supernatant!
CAUTION Do not discard beads!
CAUTION Be careful not to disturb the beads that contain DNA targets.
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.
SAFE STOP: Samples can be stored at –20°C.
Be careful not to disturb the beads.
CAUTION Be aware that bead drying starts with removing the supernatant, as the beads are not covered with ethanol anymore. Therefore start timer (3 minutes and 30 seconds) once you have removed the supernatant.
Carefully remove and discard the supernatant with a p200 pipette.
With a p10 or p20 pipette, remove residual traces of ethanol.
270 bp
320 bp
PCR Enrichment of Adaptor-ligated DNA
What type of indexing do you intend to use?
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1)
4.1.1A Prepare the following PCR reaction in a sterile PCR tube
NEBNext Multiplex Oligos for Illumina (Index Primers Set 2)
NEBNext Multiplex Oligos for Illumina (Index Primers Set 3)
NEBNext Multiplex Oligos for Illumina (Index Primers Set 4)
Adaptor Ligated DNA Fragments (Step 3 result)
15 μl
25 μl
(blue) Index i7 Primer
5 μl
(blue) Index i5 Primer
Total volume
40 μl
4.1.3 PCR Amplification
Place the tube in a thermal cycler and perform PCR amplification using the following PCR cycling conditions
4.1.1B Prepare the following PCR reaction in a sterile PCR tube
Index Primer Mix
CYCLE STEP
TEMP
Initial Denaturation
TIME
CYCLES
98°C
30 seconds
Denaturation
10 seconds
cycles
Annealing/Extension
65°C
75 seconds
Final Extension
5 minutes
Hold
4°C
∞
What input amount did you start the library prep with?
PCR cycles for standard library prep to generate a yield of ~100 ng (30–100 nM):
5 ng
10 ng
50 ng
100 ng
500 ng
1 μg
1 ng
0.5 ng
PCR cycles for target enrichment library prep to generate a yield of ~1 μg:
* According to your input and yield requirements!
Place the tube in a thermal cycler and perform PCR amplification using the following PCR cycling conditions.
Which oligos are you using?
Single Index
Dual Index
Unique Dual Index
NEBNext Multiplex Oligos for Illumina (96 Index Primers)
4.1.2 Mix PCR Reaction
Set a pipette to: 40 μl
Mix by pipetting 15 times, then quick spin.
NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1)
NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 2)
(blue) Index Primer
(blue) Universal PCR Primer
Input DNA in the end prep reaction
#Cycles
3 - 4
6 - 7
7 - 8
9 - 10
10 - 11
4 - 5
12 - 13
14 - 15
Add the Primers to the PCR reaction
Add Primer to the PCR reaction
Quick spin the index primers, then mix by flicking 10 times, followed by a quick spin.
Mix index primers
Mix the NEBNext Ultra II Q5 Master Mix by inverting 10 times, followed by flicking 10 times, then quick spin.
Quick spin the index primer plate, then mix by vortexing 3 seconds, followed by a quick spin.
Mix index primer plate
Mix NEBNext Ultra II Q5 Master Mix
Cleanup of PCR Reaction
Transfer 30 μl of library to a new PCR tube.
5.11 Separate beads from the eluate
Place the tube/plate on a magnetic stand and wait for 5 minutes to separate the beads from the eluate.
5.10 Mix
5.9 Elution of DNA
Remove the tube/plate from the magnetic stand and elute the DNA from the beads by adding 33 μl of 0.1X TE.
5.8 Dry the beads
5.6 First ethanol wash
5.5 Discard supernatant
5.4 Separate beads from supernatant
5.3 Incubate
Mix Beads with PCR Reaction
Set a pipette to: 80 μl
5.2 Add Beads to PCR Reaction
Add 45 μl of resuspended beads to the 50 μl of PCR Reaction.
5.1 Vortex Beads
SAFE STOP: DNA libraries can be stored at –20°C.
5.7 Second ethanol wash
Thank you for choosing
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