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  • NEBNext® dsDNA Fragmentase®

    Description

    NEBNext®  dsDNA Fragmentase generates dsDNA breaks in a time-dependent manner to yield 50–1,000 bp DNA fragments depending on reaction time (1). NEBNext dsDNA Fragmentase contains two enzymes, one randomly generates nicks on dsDNA and the other recognizes the nicked site and cuts the opposite DNA strand across from the nick, producing dsDNA breaks. The resulting DNA fragments contain short overhangs, 5´-phosphates, and 3´-hydroxyl groups. The random nicking activity of NEBNext dsDNA Fragmentase has been confirmed by preparing libraries for next-generation sequencing. A comparison of the sequencing results between gDNA prepared with NEBNext dsDNA fragmentase and with mechanical shearing demonstrates that the NEBNext dsDNA Fragmentase does not introduce any detectable bias during the sequencing library preparation and no difference in sequence coverage is observed using the two methods (2).

    (1). Patent pending.
    (2). Unpublished observations.
     
    Reaction Definition:  One reaction is defined as the amount of NEBNext dsDNA Fragmentase required to convert 1 µg of purified HeLa cell gDNA in 20 µl of 1X NEBNext dsDNA Fragmentase Reaction Buffer v2 into short (100–300 bp) DNA fragments in 30 minutes at 37°C.



    Figure 1: Fragmentation of E. coli gDNA. E. coli gDNA was fragmented with NEBNext dsDNA Fragmentase for the indicated times and purified on MinElute® columns.
    Figure 2: Fragmentation of 5,10 and 20 kb amplicons with NEBNext dsDNA Fragmentase.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Magnesium Chloride (MgCl2) (200 mM)
    NEBNext dsDNA Fragmentase Reaction Buffer v2-2010X

    Advantages and Features

    Applications

    • Generation of dsDNA fragments for sequencing on next generation sequencing platforms
    • Generation of dsDNA fragments for libraries

    Properties and Usage

    Storage Temperature

    -20°C

    Quality Control

    Quality Assurance Statement

    • Free of detectable protease and phosphatase activity.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Heat Inactivation: 65°C for 15 minutes in the presence of 50mM DTT.

    Supporting Documents

    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.

    Datacards

    The Product Summary Sheet, or Data Card, 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 the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Is NEBNext dsDNA Fragmentase cleavage random?
    2. Why has the buffer been changed?
    3. Is there any change to the Fragmentase enzyme?
    4. If I can’t make this change to the new buffer right now, can I still obtain the original Fragmentase buffer?
    5. What is the difference between Fragmentase Reaction Buffer (#B0348) and Fragmentase Reaction Buffer v2 (#B0349)?
    6. What other components are now included along with the dsDNA Fragmentase in M0348?
    7. Where are the protocols and application notes for AT- or GC-rich genomes, and for PCR products with dsDNA Fragmentase?
    8. Why is NEBNext E. coli DNA Ligase for Fragmentase no longer included or recommended for this application?
    1. Digestion with NEBNext dsDNA Fragmentase (M0348)
    If you generate more plasmid DNA by bacterial transformation, we recommend isolating the plasmid DNA using an endotoxin-free plasmid prep kit prior to transfection into mammalian cells.