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  • Preparation of Adaptor Ligated DNA (E6270)

    Protocol

    1. Add the following to the microfuge tube:
        For 10 ng For 100 ng – 1 µg
      Sterile H2O 18 µl 3 µl
      T4 DNA Ligase Buffer for Ion Torrent 10 µl 10 µl
      Bst 2.0 WarmStart DNA Polymerase 1 µl 1 µl
      NEBNext DNA Library Adaptors for Ion Torrent 5 µl 20 µl
      T4 DNA Ligase 6 µl 6 µl
      Total volume 40 µl 40 µl


    2. The total volume in the microfuge tube should be 100 µl. Mix the contents by pipetting up and down several times.

    3. Incubate in a thermal cycler for 15 minutes at 25°C, followed by 5 minutes at 65°C, hold at 4°C.
    A precipitate can form upon thawing of the NEBNext Hot Start HiFi PCR Master Mix. To ensure optimal performance, place the master mix at room temperature while performing size selection/cleanup of adaptorligated DNA. Once thawed, gently mix by inverting the tube several times.
     
    If performing size selection with beads, proceed directly to size selection using AMPure XP Beads. If using E-gel or agarose gel for size selection, proceed to Cleanup of Adaptor Ligated DNA before proceeding to gel-based size selection.