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  • Preparation of Adaptor Ligated DNA (E6285)


    1. Add the following to the microfuge tube:
        For 10 ng   For 100 ng – 1 μg
      Bst DNA Polymerase, Large Fragment 1 µl 1 µl
      Sterile H2O 9 µl 1 µl
      T4 DNA Ligase Buffer for Ion Torrent 4 µl 4 µl
      NEBNext DNALibrary Adaptors for Ion Torrent 2 µl 10 µl
      T4 DNA Ligase 4 µl 4 µl
      Total volume 20 µl 20 µl

    2. The total volume in the microfuge tube should be 40 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, store on ice.

    4. Add 5 μl Stop Buffer, vortex and pulse-spin.

    If performing bead based size selection, add 60 μl of 0.1X TE and proceed directly to the bead-based size selection protocol. If using E-gel or agarose gel for size selection,clean up adaptor ligated DNA before proceeding to size selection.