New England BioLabs NEBNext Ultra II User manual

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INSTRUCTION MANUAL
NEBNext®Ultra™ II End Repair/dA-Tailing Module
NEB #E7546S/L 24/96 reactions
Version 3.0_3/20
Table of Contents
Protocol .....................................................................................................................................................................................2
Kit Components ........................................................................................................................................................................2
Revision History .......................................................................................................................................................................3
The NEBNext Ultra II End Repair/dA-Tailing Module Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7546S) and 96 reactions (NEB #E7546L). All reagents
should be stored at –20°C. Colored bullets represent the color of the cap of the tube containing the reagent.
• (green) NEBNext Ultra II End Prep Reaction Buffer
• (green) NEBNext Ultra II End Prep Enzyme Mix
The NEBNext Ultra II End Repair/dA-Tailing Module is Designed for use with the Following:
NEBNext Ultra II Ligation Module (NEB #E7595)
NEBNext Ultra II Q5® Master Mix (NEB #M0544)
NEBNext Singleplex or Multiplex Oligos for Illumina®
(NEB #E7350, #E7335, #E7500, #E7600 or #E7535)
Applications
The NEBNext Ultra II End Repair/dA-Tailing Module is optimized to convert 500 pg-1 µg of fragmented DNA to repaired DNA having 5´
phosphorylated, 3´ dA-tailed ends.
Lot Control: The lots provided in the NEBNext Ultra II End Repair/dA-Tailing Module for Illumina are managed separately and qualified
by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet
stringent criteria in the additional quality controls listed on each individual component page.
Functionally Validated: Each set of reagents is functionally validated together through construction and sequencing of an indexed DNA
library on the Illumina sequencing platform.
For larger volume requirements, customized and bulk packaging is available
by purchasing through the OEM/Bulks department at NEB. Please contact [email protected] for further information.

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Protocol
Symbols
This caution sign 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.
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.
Colored bullets indicate the cap color of the reagent to be added.
Starting Material: 500 pg–1 µg fragmented DNA. We recommend 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, 10 mM Tris-HCl, pH 8.0 or 0.1X TE can be used.
1. NEBNext End Prep
1.1. Add the following components to a sterile nuclease-free tube:
END PREP REACTION
VOLUME
• (green) NEBNext Ultra II End Prep Reaction Buffer
7 µl
• (green) NEBNext Ultra II End Prep Enzyme Mix
3 µl
Fragmented DNA
50 µl
Total Volume
60 µl
1.2. 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: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
1.3. Place in a thermal cycler, with the heated lid set to ³ 75°C, and run the following program:
30 minutes @ 20°C
30 minutes @ 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.
1.4. Proceed directly to NEBNext Ultra II Ligation Module (NEB #E7595)
Kit Components
NEB #E7546S Table of Components
NEB #
PRODUCT
VOLUME
E7646A
NEBNext Ultra II End Prep Enzyme Mix
0.072 ml
E7647A
NEBNext Ultra II End Prep Reaction Buffer
0.168 ml
NEB #E7546L Table of Components
NEB #
PRODUCT
VOLUME
E7646AA
NEBNext Ultra II End Prep Enzyme Mix
0.288 ml
E7647AA
NEBNext Ultra II End Prep Reaction Buffer
0.672 ml

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Revision History
REVISION #
DESCRIPTION
DATE
1.0
N/A
8/15
2.0
Create "Kit Component – Table of Components" for small and large
size kits. Delete individual component information pages.
4/18
3.0
New Format Applied.
3/20
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.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information about commercial
rights, please email us at [email protected] While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
ILLUMINA®is a registered trademark of Illumina, Inc.
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