Product Class: Kit

NEBNext® Multiplex Oligos for Illumina® (96 Index Primers)

Product Introduction

NEBNext® Multiplex Oligos provide adaptors and primers to enable high yield multiplex Illumina® library production. The unique hairpin loop structure of the NEBNext Adaptor minimizes adaptor-dimer formation, and NEBNext index PCR primers enable index incorporation during library amplification.

The 96 8-base index primers included in this kit are pre-mixed with the universal primer and are packaged in a single-use 96-well plate with a pierceable foil seal.

For additional information on available indices and formats for multiplexing, please refer to the NEBNext Multiplex Oligos Selection Chart.

  • High efficiency adaptor ligation
  • Minimized adaptor-dimer formation
  • Includes index primers for library multiplexing
  • 96-well plate format with pierceable foil seal
Catalog # Size Concentration
E6609S 96.0 reactions
E6609L 384.0 reactions

Product Information

Description

Incorporating a novel hairpin loop structure, the NEBNext Adaptor ligates with increased efficiency to end-repaired, dA-tailed DNA. The loop contains a U, which is removed by treatment with USER® Enzyme (a mix of UDG and Endo VIII), to open up the loop and make it available as a substrate for PCR. During PCR, barcodes can be incorporated by use of the NEBNext index primers, thereby enabling multiplexing.

The 96 8-base index primers included in this kit are pre-mixed with the universal primer and are packaged in a single-use 96-well plate with a pierceable foil seal.

NEBNext Oligos can be used with NEBNext products, and with other standard Illumina-compatible library preparation protocols.

Additional single- or dual-barcode primer options are available, in 12- and 96- index formats (NEB #E7335, NEB #E7500, NEB #E7710, NEB #E7730, NEB #E7600). A methylated version of the NEBNext Adaptor is also available for use with bisulfite sequencing protocols (NEB #E7535).

Figure 1: Workflow demonstrating the use of NEBNext Multiplex Oligos for Illumina (96 Index Primers)

Figure 1: Workflow


This product is related to the following categories:
NEBNext® Multiplex Oligos (Adaptors & Primers) Products,
Next Generation Sequencing Library Preparation Products,
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E6609S     -20    
      USER® Enzyme E6610AVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Index/Universal Primer Mix (1-96) E6611AVIAL -20 1 x 10 μl/well 10 µM
      NEBNext® Adaptor for Illumina® E6612AVIAL -20 1 x 0.96 ml 15 µM
  • E6609L     -20    
      USER® Enzyme E6610AAVIAL -20 2 x 0.576 ml Not Applicable
      NEBNext® Index/Universal Primer Mix (1-96) E6611AVIAL -20 4 x 10 μl/well 10 µM
      NEBNext® Adaptor for Illumina® E6612AVIAL -20 4 x 0.96 ml 15 µM

Properties & Usage

Materials Required but not Supplied

  • Enzymes and buffers appropriate for DNA, ChIP or mRNA Library Preparation (NEB #E7645, E7595, E7420, E7530, E7370, E7445, E6040, E6110, E6240, E6056, E6000, E6200, E6100)
  • SPRIselect or AMPure® XP Beads (Beckman Coulter, Inc.)
  • DNA LoBind® Tubes (Eppendorf®)/PCR Plates
  • Magnetic Stand
  • Bioanalyzer® (Agilent Technologies, Inc.)
  • Freshly Prepared 80% Ethanol

Please Refer to the Kit Specific Protocol for using the NEBNext Multiplex Oligos for Illumina:
The following kits are designed for use with the NEBNext Multiplex Oligos for Illumina:

  • #E7760, NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
  • #E7765, NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads
  • #E7770, NEBNext Ultra II RNA library Prep Kit for Illumina
  • #E7775, NEBNext Ultra II RNA Library Prep with Sample Purification Beads
  • #E7805, NEBNext Ultra II FS DNA Library Prep Kit for Illumina
  • #E6177, NEBNext Ultra II FS DNA Library Prep Kit with Sample Purification Beads
  • #E7645, NEBNext Ultra II DNA Library Prep Kit for Illumina
  • #E7103, NEBNext Ultra II DNA Library Prep with Sample Purification Beads
  • #E7595, NEBNext Ultra II Ligation Module
  • #E7420, NEBNext Ultra Directional RNA Library Prep Kit for Illumina
  • #E7530, NEBNext Ultra RNA Library Prep Kit for Illumina
  • #E7370, NEBNext Ultra DNA Library Prep Kit for Illumina
  • #E7445, NEBNext Ultra Ligation Module
  • #E6040, NEBNext DNA Library Prep Master Mix Set for Illumina
  • #E6240, NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina
  • #E6056, NEBNext Quick Ligation Module

Protocols, Manuals & Usage

Protocols

  1. Setting up the PCR Reactions < 96 Samples and 96 Samples - 96 Single Index Kit (E6609)
  2. Index Pooling Guidelines - 96 Single Index Kit (E6609)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Are NEBNext adaptors and primers for Illumina validated in Next Generation Sequencing Workflows?
  2. Are the NEBNext Oligos for Illumina compatible with single read and paired end sequencing?
  3. What is the concentration of the adaptor and primers in the NEBNext Multiplex Oligos kits?
  4. Can I use the 96-well primer plate to do my PCR reactions when using the NEBNext® Multiplex Oligos for MGI® (Dual Index Primer Pairs Set 1) (NEB #E9640)?
  5. How many reactions worth of primer are in each well?
  6. Do you have a Sample Sheet with the index sequences that can be used in the Illumina Experiment Manager (IEM)? 
  7. Is the adaptor methylated?
  8. Is the USER enzyme in our NEBNext kits identical in concentration to the standalone product?
  9. Is USER enzyme required for library preparation using NEBNext adaptor and primers for Illumina?
  10. Can USER Enzyme be purchased separately?
  11. Are these reagents available in a customized format, larger format or 96 well formats?
  12. How do I use the NEB provided Sample Sheet for my specific instrument?
  13. What sequences need to be trimmed for NEBNext libraries that are sequenced on an Illumina instrument?

Troubleshooting

NEBNext® DNA Library Prep Reagent Set for Illumina®

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Trademarks

NEW ENGLAND BIOLABS® and NEBNEXT® are registered trademarks of New England Biolabs, Inc.
USER™ is a trademark of New England Biolabs, Inc.
AMPURE® is a registered trademark of Beckman Coulter, Inc.
BIOANALYZER® is a registered trademark of Agilent Technologies, Inc.
EPPENDORF® and LOBIND® are registered trademarks of Eppendorf AG.
ILLUMINA®, HISEQ® and MISEQ® are registered trademarks of Illumina, Inc.

© Copyright 2016, New England Biolabs, Inc; all rights reserved.

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