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  • Fragmentation and End Repair of DNA Protocol (E6285)

    Overview

    Starting Material: 10 ng–1 μg of DNA.

    Note: For use with the Ion Xpress™ Barcode Adapters 1-16 Kit, a minimum of 100 ng starting material is recommended. Lower amounts may cause adaptor concatamerization.

    DNA quality and composition can affect fragmentation. Be sure to use the highest quality DNA.

    Note: For GC Rich content, add 1 μl of MgCl2 to the reaction. If fragmentation is insufficient, add additional 10 mM (1 μl) of MgCl2 to reach desired fragment size. See Figure 1 on product page  for typical fragment size distribution.

    Protocol

    1. Mix the following components in a sterile microfuge tube on ice:
      DNA   1–15.5 μl
      NEBNext DNA Fragmentation Reaction Buffer   2 μl
      Sterile H2O   variable
      -----------------------------------------------------------------------
      Total volume   18.5 μl

    2. Vortex for 3 seconds, pulse spin and place on ice.

    3. Vortex the vial of NEBNext DNA Fragmentation Master Mix for 3 seconds and pulse spin to collect liquid from the sides of the tube.

    4. Add 1.5 μl of NEBNext DNA Fragmentation Master Mix to the microfuge tube, vortex for 3 seconds and pulse spin.

      Note: For DNA with an AT content ≥ 70%, add 0.75 μl of NEBNext Fragmentation Master Mix and 0.75 μ l sterile H2O.

    5. Incubate in a thermal cycler for 20 minutes at 25°C, followed by 10 minutes at 70°C.

    6. Pulse spin the microfuge tube and return to ice.