INSTRUCTION MANUAL
NEBNext UltraExpress™ FS DNA Library Prep Kit
NEB #E3340S/L

24/96 reactions
Version 2.0_4/24

Table of Contents
Overview .................................................................................................................................................................................. 2
Protocol .................................................................................................................................................................................... 3
Fragmentation/End Prep ..................................................................................................................................................... 3
Adaptor Ligation ................................................................................................................................................................ 4
PCR Enrichment of Adaptor-ligated DNA ............................................................................................................................ 5
Phased Bead Cleanup of PCR Reaction ................................................................................................................................ 6

Appendix A: Recommendations for customized fragmentation time and/or cleanup to alter library size distributions ................ 8
Appendix B: Recommendations for customized cycling per input ............................................................................................. 9
Kit Components ...................................................................................................................................................................... 10
Revision History ..................................................................................................................................................................... 10

The Library Kit Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E3340S) and 96 reactions (NEB #E3340L).
All reagents should be stored at –20°C. Colored bullets represent the color of the cap of the tube containing the reagent.

•
•
•
•
•
•

(yellow) NEBNext UltraExpress FS Enzyme Mix(yellow) NEBNext UltraExpress FS Reaction Buffer(red) NEBNext UltraExpress Ligation Master Mix(blue) NEBNext® MSTC™ High Yield Master Mix(white) TE Buffer (1X)
(white) NEBNext Bead Reconstitution Buffer

Required Materials Not Included• 80% Ethanol (freshly prepared)• Nuclease-free Water• DNA LoBind® Tubes (Eppendorf® #022431021)• DNase-, RNase-free PCR strip tubes• NEBNext Singleplex or Multiplex Oligos for Illumina®
• NEBNext Multiplex Oligos options can be found at www.neb.com/oligos. Alternatively, customer supplied adaptor and primers
can be used; please see information in link below:
https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb
Note: this protocol is only for use with non-indexed adaptors• SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)• Magnetic rack/stand (NEB #S1515, Alpaqua® cat. #A001322, or equivalent)• Vortex• Thermal cycler

1• Bioanalyzer® or TapeStation® (Agilent® Technologies, Inc.) and associated reagents and consumables• NanoDrop® or Lunatic® (Unchained Labs®) and associated consumables• NEBNext Adaptor for Illumina Overview
The following sequences are used for adaptor trimming of NEBNext adaptors for Illumina:• Read 1 AGATCGGAAGAGCACACGTCTGAACTCCAGTCA• Read 2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

Overview
The NEBNext UltraExpress FS DNA Library Prep Kit contains the enzymes and buffers required to rapidly convert 10–200 ng of intact DNA
into high-quality libraries for sequencing on the Illumina platform. The fast and simple workflow features reduced cleanup steps, minimal handson time, no adaptor dilutions, and a single PCR cycling condition across the entire input range. The protocol also allows for library preparation ina single tube, minimizing plastic consumables waste. In addition to the standard protocol, the appendices detail: (A) fragmentation times andcleanup conditions for altering library size distribution and (B) cycling recommendations for optimizing library yields from varying DNA input
amounts, if required.

Note: The UltraExpress FS DNA Library Prep Kit is not compatible with bisulfite or enzymatic conversion workflows and
FFPE DNA.
Each kit component must pass rigorous quality control standards and for each new lot the entire set of reagents is functionally validated
together by construction and sequencing of indexed libraries on an Illumina sequencing platform.
Where larger volumes, customized or bulk packaging are required, we encourage consultation with the Customized Solutions team at NEB.
Please complete the NEB Custom Contact Form at www.neb.com/CustomContactForm to learn morePlease refer to the product page on
NEB.com for FAQs about this product.
Figure 1. Workflow demonstrating the use of NEBNext UltraExpress FS DNA Library Prep Kit

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Protocol
Symbols

This is a point where you can safely stop the protocol.

This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable,
like the amount of input DNA.

Colored bullets indicate the cap color of the reagent to be added to a reaction.
Starting Material: 10–200 ng of intact DNA. If the DNA volume is less than 15 µl, add TE Buffer (1X) to a final volume of 15 µl.
Alternatively, samples can be diluted with 10 mM Tris-HCl, pH 8.0; low EDTA TE; 0.1X TE; or Nuclease-free water.

1. Fragmentation/End Prep
Note:
(1)
(2)
(3)
(4)

Ensure that the FS Reaction Buffer is completely thawed. If precipitate is seen in the buffer, pipette up and down several
times to break it up, vortex to mix, and place on ice prior to use.
It is important to vortex the FS enzyme mix prior to use for optimal performance.
See Figure 1.1 for typical fragment, library, and insert size distributions.
Fragmentation occurs during the 37°C incubation step in a time-dependent manner; optionally, see Appendix A for varying
fragmentation time and library cleanup conditions to alter library size distributions.

1.1.

Vortex the UltraExpress FS Enzyme Mix and Reaction Buffer 5–8 seconds prior to use and place on ice. A master mix of the
enzyme mix and buffer may be prepared immediately prior to use.

1.2.

Add the following components to a sterile nuclease-free tube on ice:

COMPONENTVOLUME

Intact DNA*

15 µl

• (yellow) NEBNext UltraExpress FS Reaction Buffer• (yellow) NEBNext UltraExpress FS Enzyme Mix

4 µl

Total Volume

20 µl

1 µl

* Intact gDNA, cDNA, plasmids, and amplicons

1.3.

Vortex the reaction for 5 seconds and briefly spin down . A small number of bubbles in the reaction will not inhibit performance.

1.4.

Place in a thermal cycler, with the heated lid set to ≥ 75°C or on, and run the following program:
20 minutes at 37°C
15 minutes at 65°C
Hold at 4°C

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.

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Figure 1.1: Example of fragment, library, and insert size distributions for 10, 50, 100 and 200 ng Human gDNA
(NA19240) inputs following 20 minutes of enzymatic fragmentation using the NEBNext UltraExpress FS DNA
Library Prep Kit.

(A) TapeStation® HS D1000 ScreenTape® traces of end prepped fragments cleaned up with 2X beads priorto adaptor ligation and final libraries made from Human gDNA (NA19240). (B) Graphical depiction oflibrary insert size distributions as determined by Picard CollectInsertSizeMetrics tool.

2. Adaptor Ligation
2.1.

Add the following components directly to the FS Reaction Mixture:
COMPONENT*VOLUMEFS Reaction Mixture (Step 1.4. in Section 1)

20 µl• (red) NEBNext Adaptor for Illumina*• (red) NEBNext UltraExpress Ligation Master Mix**

2 µl
20 µl

Total Volume

42 µl

The NEBNext adaptor is provided in NEBNext oligos kit. NEB has several oligo kit options,which are supplied separately from the library prep kit. www.neb.com/oligos** Mix the UltraExpress Ligation Master Mix by pipetting up and down several times prior to adding
to the reaction.

Note: Do not premix the NEBNext UltraExpress Ligation Master Mix and Adaptor prior to use in the Adaptor
Ligation Step.
2.2.

Set a 100 µl pipette to 40 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 UltraExpress 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 number of bubbles will not interfere with performance).

2.3.

Incubate at 20°C for 15 minutes in a thermal cycler with the heated lid off.
Note: Steps 2.4. and 2.5. are only required for use with NEBNext Adaptor (loop adaptor). USER® enzyme can be found in
NEBNext Multiplex Oligos for Illumina sets.

2.4.

Add 3 µl of • (red) USER Enzyme to the ligation mixture from Step 2.3.

2.5.

Mix well and incubate at 37°C for 5 minutes in a thermal cycler with the heated lid set to ≥ 47°C or on.

Samples can be stored at –20°C overnight.

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3. PCR Enrichment of Adaptor-ligated DNA

Use Option A (3.1.1A): for any NEBNext Index Primers where the forward and reverse primers are supplied separately in tubes. Primers
are supplied at 10 μM.
Use Option B (3.1.1B): for any NEBNext Index Primers where index primers are supplied with the forward and reverse primers (i5 and i7)
premixed in a 96-well plate format. These primers are supplied at a 10 μM combined concentration (5 μM each).

3.1. PCR Amplification
3.1.1.

Add the following components to a sterile strip tube:
3.1.1A. Forward and Reverse Primers not already combined (Option A)
COMPONENTVOLUME

Adaptor Ligated DNA Fragments (Step 2.5.)45 µl• (blue) NEBNext MSTC High Yield Master Mix• (blue) Index Primer/i7 Primer*,**• (blue) Universal PCR Primer/i5 Primer*,**45 µl

Total Volume

100 µl5 µl
5 µl

3.1.1B. Forward and Reverse Primers already combined (Option B)
VOLUMECOMPONENT
Adaptor Ligated DNA Fragments (Step 2.5.)45 µl• (blue) NEBNext MSTC High Yield Master Mix

45 µl

Index/Universal Primer*,**

10 µl

Total Volume

100 µl* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding
NEBNext Multiplex Oligos Kit manual for determining valid barcode combinations.
** Use only one i7 primer/index primer per sample. Use only one i5 primer (or the universal primer for
single index kits) per sample.

3.1.2.

Set a 100 µl or 200 µl pipette to 90 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.

3.1.3.

Place the tube in a thermal cycler with the heated lid set to 105°C or on and perform PCR amplification using the following PCR
cycling conditions:
CYCLE STEP

TEMP

TIME

CYCLES

Initial Denaturation
Denaturation

98°C
98°C

30 seconds
10 seconds

1

Annealing/Extension

65°C

75 seconds

Final Extension

65°C

Hold

4°C

5 minutes∞

6*1* PCR amplification with 6 cycles can generate sufficient library yields for sequencing from DNA inputs
ranging from 10–200 ng and allows for one PCR condition across the entire input range.

Note:(1) For DNA inputs higher than 100 ng, 6 cycles of PCR can result in amplification beyond the linear phase during thelate cycles. This can cause inaccurate library quantification on migration-based methods like Bioanalyzer and
TapeStation (See Figure 4.1). In this case, we recommend using Nanodrop or Lunatic for library quantification.
(2) The adaptor amount and PCR cycle numbers were validated using high quality genomic DNA and NEBNext adaptor
for Illumina. For other DNA input types, we recommend optimization of adaptor amount and PCR cycles.
(3) For more specific library yield requirements, you may choose to follow Appendix B for recommended number of
PCR cycles.

5(4) Take out NEBNext Bead Reconstitution Buffer and bring up to room temperature prior to Phased Bead Cleanup.
Allow the buffer (and beads if using AMPure XP) to warm to room temperature for at least 30 minutes before use.
Proceed to Cleanup of PCR Amplification in Section 4.

4. Phased Bead Cleanup of PCR Reaction
Note: The SPRIselect/Ampure Beads ratios recommended in this manual have been experimentally optimized for every step;
this is critical since buffer compositions differ between steps and across protocols. Please adhere to these guidelines andnot those recommended by other sources or for other kits. If using Ampure Beads, remove from 4°C and keep at room
temperature for 30 minutes prior to use.
4.1.

Vortex SPRIselect/Ampure Beads to resuspend. Thaw and mix NEBNext Bead Reconstitution Buffer at room temperature andafterwards it may be stored in a sealed container at room temperature for future use.

4.2.

Add 70 μl (0.7X) of resuspended beads to the 100 μl PCR reaction. 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.

4.3.

Incubate samples on bench top for at least 5 minutes at room temperature.

4.4.

Place the tube/ plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin thesample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

4.5.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that
contain DNA targets (Caution: do not discard the beads).

4.6.

Remove the tube/ plate from the magnetic stand (Note: no ethanol wash at this step). Add 100 μl of 0.1 X TE (dilute 1X TE
Buffer 1:10 in water) to resuspend the beads. Mix well by pipetting up and down at least 10 times.

4.7.

Add 80 μl (0.8X) of NEBNext Bead Reconstitution Buffer to the 100 μl of resuspended beads. 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.

4.8.

Incubate samples on bench top for at least 5 minutes at room temperature.

4.9.

Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin thesample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

4.10.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that
contain DNA targets (Caution: do not discard the beads).

4.11.

Add 200 μl of freshly prepared 80% ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for
30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

4.12.

Repeat Step 4.11. once for a total of two washes. 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.

4.13.

Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. 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 tocrack, they are too dry.

4.14.

Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 0.1X TE (dilute 1X TE
Buffer 1:10 in water).

4.15.

Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes 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.

4.16.

Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 μl to a new PCR tube andstore at –20°C.

4.17.

Check the size distribution on an Agilent Bioanalyzer High Sensitivity DNA chip or TapeStation D1000 HS ScreenTape. The sample
may need to be diluted before loading.

Samples can be stored at –20°C.

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Figure 4.1: Examples of libraries prepared with human DNA (NA19240).

Libraries made from (A) 10 ng and (B) 200 ng human gDNA (NA19240) with 6 PCR cycles were diluted 1:3 and run on a
TapeStation HSD1000 ScreenTape. Figure 4.1.B shows a peak above the upper marker (indicated by the red arrow) resulting from
non-linear amplification phase for the 200 ng sample at late PCR cycles. Although the molecules generated during the non-linear
amplification phase are sequencible, their different migration speed on ScreenTapes causes inaccurate quantification of the library
concentration. In this case, we recommend measuring library concentration on NanoDrop or Lunatic using 400 bp or the intended
library size to convert the concentration values to nanomolar. The following equation can be used to convert concentration fromng/μl to nM: [(Library Concentration in ng/μl) / (660 g/mol x Library Size in bp)] x 106 = Concentration in nM.
Note: If excess adaptor dimer peak is observed at 150–180 bp, an additional 0.8X bead cleanup can be performed on
individual or pooled libraries. The additional cleanup will result in a reduction in yield and slightly larger libraries,
which will not affect sequencing results.

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Appendix A
Recommendations for customized fragmentation time and/or cleanup to alter
library size distributions
The standard protocol described elsewhere in this Instruction Manual is designed to offer fast, streamlined library prep. However, forcases where customization is desired, we provide the following recommendations for customized fragmentation times at 37°C and/or
phased final library cleanup conditions for varying final library size distributions.
Note: Phased cleanups with less than 0.5X bead addition result in decreased library yields. The following recommendations
are for libraries made from DNA > 1 kb in size or intact gDNA inputs of at least 100 ng following the 6-cycle PCR protocol(Step 3.1.3.). To apply custom phased cleanups on inputs of 10–100 ng, we recommend additional PCR cycling prior tocleanup (see Appendix B).
Table A1. Approximate size distributions for libraries and inserts with custom fragmentation time and/or phased cleanups.
The approximate library and insert sizes were determined using TapeStation traces and sequence data analysis with the Picard
CollectInsertSizeMetrics.
FRAGMENTATIONTIME(MINUTES)205PHASED CLEANUP REAGENT VOLUMESTO BE ADDED (µl)
BEAD
ADDITION(STEP 4.2.)

APPROXIMATELIBRARY PARAMETERS

BEAD RECONSTITUTION LIBRARY SIZE
INSERT SIZE PEAK INSERTBUFFER ADDITION
DISTRIBUTION DISTRIBUTIONSIZE(STEP 4.7.)
(bp)
(bp)
(bp)

70 (0.7X)

80 (0.8X)

200–72080–600125

60 (0.6X)

70 (0.7X)

220–770

100–650175

50 (0.5X)

60 (0.6X)

250–820

130–700250

40 (0.4X)

50 (0.5X)

350–920

230–800375

70 (0.7X)

80 (0.8X)

200–82080–700150

60 (0.6X)

70 (0.7X)

220–850

100–730200

50 (0.5X)

60 (0.6X)

250–920

130–800275

40 (0.4X)

50 (0.5X)

350–1,000

230–880400

Figure A1. Example of library and insert size distributions for 100 ng Human gDNA (NA19240) input following
5- or 20-minute enzymatic fragmentation and varying phased cleanup conditions.

TapeStation HS D5000 ScreenTape traces of (A) final libraries made from 100 ng Human gDNA (NA19240) fragmented for
20 minutes or 5 minutes and cleaned using a standard or custom phased cleanup. The first TapeStation lane on the left is thelibrary trace for the standard fragmentation and cleanup condition. (B) Graphical depiction of library insert size distribution
as determined by Picard CollectInsertSizeMetrics tool.

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Appendix B
Recommendations for customized cycling per input
The standard protocol described elsewhere in this Instruction Manual is designed to offer fast, streamlined library prep. However, forcases where customization is desired, we provide the following recommendations for customized PCR cycling per input.
Follow recommended PCR cycle numbers for different input amounts provided inTable B1 to obtain 100 ng library yield using undiluted NEBNext Adaptor for Illumina.
Table B1. For a library yield of 100 ng.
INPUT DNA IN
FRAGMENTATION/
END PREP REACTION# OF CYCLES REQUIREDFOR STANDARD LIBRARY PREP
Yield > 100 ng (> 15 nM final cleanup 30 µl library)≥ 100 ng

3–4

50 ng

5–6

10 ng

6–7

Follow recommended PCR cycle numbers for different input amounts provided inTable B2 to reach 1µg library yield using undiluted NEBNext Adaptor for Illumina.
Table B2. For a library yield of 1 µg.
INPUT DNA IN
FRAGMENTATION/
END PREP REACTION# OF CYCLES REQUIREDFOR TARGET ENRICHMENT LIBRARY PREP:
Yield ~1 µg (~150 nM final cleanup 30 µl library)≥ 100 ng

5–6

50 ng

7–8

10 ng

9–10

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Kit Components
NEB #E3340S Table of Components
NEB #E3347A
E3344A
E3343ACOMPONENTVOLUMETE Buffer (1X)
NEBNext UltraExpress FS Reaction Buffer

0.72 ml
0.096 ml

NEBNext UltraExpress FS Enzyme Mix

0.024 ml

E3345A

NEBNext UltraExpress Ligation Master Mix

0.48 ml

E3346A

NEBNext MSTC High Yield Master Mix

1.08 ml

E3348A

NEBNext Bead Reconstitution Buffer

1.92 ml

NEB #E3340L Table of Components
NEB #COMPONENTVOLUME

E3347AATE Buffer (1X)

2.88 ml

E3344AA

NEBNext UltraExpress FS Reaction Buffer

0.384 ml

E3343AA
E3345AA

NEBNext UltraExpress FS Enzyme Mix
NEBNext UltraExpress Ligation Master Mix

0.096 ml
2 x 0.96 ml

E3346AA

NEBNext MSTC High Yield Master Mix

4.32 ml

E3348AA

NEBNext Bead Reconstitution Buffer

7.68 ml

REVISION #DESCRIPTION

DATE

1.0

N/A
Updated figure legend to match graph. Updated
header and footer with new logo.

12/23

Revision History

2.0

4/24

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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AGILENT®, BIOANALYZER®, TAPESTATION® and SCREENTAPE® are registered trademarks of Agilent Technologies, Inc.
ALPAQUA® is a registered trademark of Alpaqua Engineering, LLC.
BECKMAN COULTER® and AMPURE® are registered trademarks of Beckman Coulter, Inc. SPRISELECT™ is a trademark of Beckman Coulter, Inc.
ILLUMINA® is registered trademarks of Illumina, Inc.
EPPENDORF® and LOBIND® are registered trademarks of Eppendorf SE.
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