New England Arbors NEBNext Ultra E7442L User manual

1
INSTRUCTION MANUAL
NEBNext®Ultra™End Repair/dA-Tailing Module
NEB #E7442L 96 reactions
Version 4.0_6/22
Table of Contents
Protocol............................................................................................................................................................................2
Kit Components................................................................................................................................................................2
Revision History...............................................................................................................................................................3
The NEBNext Ultra End Repair/dA-Tailing Module Includes:
The volumes provided are sufficient for preparation of up to 96 reactions (NEB #E7442L). All reagents should be stored at –20°C.
Colored bullets represent the color of the cap of the tube containing the reagent.
(green) NEBNext End Repair Reaction Buffer
(green) NEBNext End Prep Enzyme Mix
The NEBNext Ultra End Repair/dA-Tailing Module is Designed for use with the Following:
NEBNext Multiplex Oligos for Illumina®(NEB.com/oligos)
NEBNext Ultra Ligation Module (NEB #E7445)
NEBNext High Fidelity 2X PCR Master Mix (NEB #M0541) or NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB #M0543)
Required Materials Not Included
•Thermal cycler
•Vortex Mixer
•Microcentrifuge
•DNase RNase free PCR strip tubes (USA Scientific®1402-1708)
Applications
The NEBNext End Prep module is optimized to convert 5 ng–1 µg of fragmented DNA to repaired DNA having 5´ phosphorylated,
dA-tailed ends.
Lot Control: The lots provided in the NEBNext Ultra End Prep Module are managed separately and qualified by additional functional
validation. Individual reagents undergo standard enzyme activity and quality control assays, and 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.

2
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 type of DNA input.
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: 5 ng–1 µg fragmented DNA
NEBNext End Prep
1. Mix the following components in a sterile, nuclease-free tube:
COMPONENT
VOLUME
(green) NEBNext End Prep Enzyme Mix
3.0 µl
(green) NEBNext End Repair Reaction Buffer
6.5 µl
Fragmented DNA
55.5 µl
Total Volume
65 µl
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.
3. Place in a thermal cycler, with the heated lid set to ≥75°C, and run the following program:
30 minutes @ 20°C
30 minutes @ 65°C
Hold at 4°C
4. Proceed directly to NEBNext Ultra Ligation Module (NEB #E7445).
Kit Components
NEB #E7442L Table of Components
NEB #
PRODUCT
VOLUME
E7371AA
NEBNext End Prep Enzyme Mix
0.288 ml
E7372AA
NEBNext End Repair Reaction Buffer
0.624 ml

3
Revision History
REVISION #
DESCRIPTION
DATE
1.0
N/A
2/13
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.
1/20
4.0
Remove small size and update protocol
6/22
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. The use of trademark symbols does
not necessarily indicate that the name is trademarked in the country where it is being read; rather, it indicates where the document was originally developed. 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 you to obtain additional third party intellectual property rights for certain applications.
B CORPORATION®is a registered trademark of B Lab IP, LLC, Inc.
ILLUMINA®is a registered trademark of Illumina, Inc.
USA SCIENTIFIC®is a registered trademark of USA Scientific, Inc.
© Copyright 2022, New England Biolabs, Inc.; all rights reserved