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  • NEBNext® mRNA Library Prep Reagent Set for 454™

    Discontinued Date

    01/01/2013
    Catalog #SizeConcentrationPriceQtyAdd to Cart
      
    Categories:
    Discontinued Products

    Description

    The NEBNext® mRNA Library Prep Reagent 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 Acrylamide10 mg/ml
    Random Primers
    Murine RNase Inhibitor
    NEBNext First Strand Synthesis Reaction Buffer5X
    NEBNext Second Strand Synthesis Enzyme Mix
    NEBNext Second Strand Synthesis Reaction Buffer10X
    NEBuffer 2-2010X
    Deoxynucleotide Solution Mix-2010 mM
    T4 DNA Polymerase-203,000 units/ml
    Adenosine 5'-Triphosphate (ATP)-20*10 mM
    T4 Polynucleotide Kinase-2010,000 units/ml
    Quick T4 DNA Ligase-20
    NEBNext Sizing Buffer1.2X
    TE Buffer
    Taq DNA Polymerase with ThermoPol® Buffer-205,000 units/ml

    * Reagents' Storage Notes

    • Adenosine 5'-Triphosphate (ATP):

      For long term storage (>30 days), store at -70°C.

    Properties and Usage

    Materials Required but not Supplied

    Agencourt® Ampure® Beads (Beckman Coulter)
    Magnetic Separation Rack (NEB #S1510)
    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)
    DNA Adaptors and Primers or Vector

    Storage Temperature

    -20°C

    Protocols

    1. mRNA Fragmentation Protocol (E6115)
    2. First Strand cDNA Synthesis (E6115)
    3. Second Strand cDNA Synthesis
    4. NEBNext End Repair and dA-Tailing (E6115)
    5. Agencourt Ampure Bead (Beckman Coulter) Preparation (E6115)
    6. NEBNext Adaptor Ligation (E6115)
    7. NEBNext Small Fragment Removal (E6115)
    8. First Strand cDNA Synthesis

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.