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  • NEBNext® Fill-in and ssDNA Isolation Module

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    The NEBNext® Fill-in and ssDNA Isolation Module has been optimized to fill in adapter sequence from a nick generated by ligation of adaptors lacking a 5´-phosphate to a DNA template coupled with ssDNA isolation. Bst DNA Polymerase, Large Fragment, recognizes the nick resulting from the ligation of unphosphorylated adapters, displaces the nicked strand and extends the 3´ end of the DNA template and fills in the adapter sequence to generate full length dsDNA. Hydrophilic Streptavidin Magnetic Beads and buffers are provided to allow binding of dsDNA fragments bearing one biotinylated adapter prior to adapter fill-in and elution of the full length, unbiotinylated ssDNA strands after adapter fill-in.

    The NEBNext® Fill-in and ssDNA Isolation Module is provided as a master mix to maximize efficiency and convenience in DNA sample preparation workflows (1,2,3). The NEBNext Fill-in and ssDNA Isolation Module has been validated by sequencing with the Roche 454 GS FLX Titanium in conjunction with the NEBNext End Repair Module, and the NEBNext Quick Ligation Module.


    Advantages and Features

    Features

    • Efficient – Enables capture of biotinylated dsDNA on Streptavidin beads. Removes nicks generated by ligation of unphosphorylated primers on 1–5 μg DNA, and enables isolation of full length ssDNA from beads.
    • Convenient – Reactions are provided in master mix format to reduce steps during DNA sample prep workflows
    • Automation Friendly

    Applications

    DNA sample preparation
    Adapter fill-in and ssDNA isolation of 1–5 μg fragmented DNA adapter ligand

    Notes

    1. Box 1: Store at -20°C
      Bst DNA Polymerase, Large Fragment 
      NEBNext Adaptor Fill-in Reaction Buffer (10X)
    2. Box 2: Store at 4°C
      Hydrophilic Streptavidin Magnetic Beads (4 mg/ml)
      NEBNext Bead Binding Buffer (2X)
      NEBNext Bead Wash Buffer (1X)

    References

    1. Maricic, T. and S. Paabo (2009). Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands. Biotechniques. 46, 51-52, 54-57.
    2. Straus, D. and F.M. Ausubel (1990). Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA. 87, 1889-1893.
    3. Zhou, X. and D.T. Wong (2007). Single nucleotide polymorphism mapping array assay. Methods Mol. Biol. 396, 295-314.

    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.
    1. NEBNext Fill-in and ssDNA Isolation Module Protocol (E6070, E6071)