NEBNext® mRNA Library Prep Master Mix Set for 454™

  • This product was discontinued on 05/12/2014
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Description

The NEBNext mRNA Library Prep Master Mix Set for 454 contains enzymes and buffers that are ideally suited for sample preparation for next-generation sequencing, and for preparation of expression libraries. Each of these components must pass rigorous quality control standards and are Lot Controlled, both individually and as a set of reagents.

Figure 1: Relative size distribution of eukaryotic mRNA fragments as seen using the Bioanalyzer 2100.
Poly (A)+ mRNA (40 ng) purified from the Universal Human Reference Total RNA (Stratagene) was fragmented in 1X NEBNext Magnesium RNA Fragmentation Buffer for 1–5 minutes at 94°C. Fragmentation Reaction was stopped in 1X NEBNext Fragmentation Stop Solution. Samples were diluted 1:10 in Nuclease-Free Water and analyzed in the Bioanalyzer 2100.
Functional Validation
Each set of reagents is functionally validated together through construction and sequencing of an RNA library on the 454 platform.

Lot Control

The lots provided 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

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBNext RNA Fragmentation Buffer10X
NEBNext RNA Fragmentation Stop Solution10X
Linear Acrylamide-2010 mg/ml
Random Primers
NEBNext First Strand Synthesis Reaction Buffer5X
NEBNext Second Strand Synthesis Enzyme Mix
NEBNext Second Strand Synthesis Reaction Buffer10X
Nuclease-free Water-20
NEBNext End Prep Enzyme Mix
NEBNext End Repair Reaction Buffer10X
Quick T4 DNA Ligase-20
TE Buffer
NEBNext Sizing Buffer1.2X

Properties and Usage

Materials Required but not Supplied

  • 3 M Sodium Acetate, pH 5.5
  • 100% Ethanol
  • 70% Ethanol
  • SuperScript® II Reverse Transcriptase (Invitrogen)
  • PCR Column Purification Kit (Qiagen or other)
  • RNeasy MinElute Columns (Qiagen or other)
  • Agencourt® Ampure® Beads (Beckman Coulter)
  • DNA Adaptors and Primers or Vector
  • Magnetic Separation Rack (NEB #S1510)

Storage Temperature

-20°C

FAQs

  1. Are NEBNext® products validated in Next Generation Sequencing workflows?

Protocols

  1. mRNA Fragmentation Protocol (E6116)
  2. First Strand cDNA Synthesis
  3. NEBNext End Repair and dA-Tailing (E6116)
  4. Agencourt Ampure Beads (Beckman Coulter) Preparation (E6116)
  5. NEBNext Adaptor Ligation (E6116)
  6. NEBNext Small Fragment Removal (E6116)
  7. Second Strand cDNA Synthesis

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Interactive Tools

Safety Data Sheet

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