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  • Digestion with NEBNext dsDNA Fragmentase (M0348)

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    Introduction

    Tip: Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 3 seconds immediately prior to use.

    For tough digestions, add 1 μl of 200 mM MgCl2 to the reaction. Additional MgCl2 can be added if necessary.

    The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.

    Protocol

    1. Vortex NEBNext dsDNA Fragmentase for 3 seconds, quick spin and place on ice.
    2. Combine the following components in a sterile PCR tube and vortex:
      DNA (5 ng–3 μg) 1–16 μl
      10X Fragmentase Reaction Buffer v2 2 μl
      Sterile Water variable
      Final Volume 18 μl
    3. Add 2.0 μl dsDNA Fragmentase and vortex the mixture for 3 seconds.
        Note: Fragmentase is very viscous and should be pipetted slowly. If the enzyme has been sitting for several minutes vortex it again before adding to the sample.
    4. Incubate at 37°C for the recommended times below to generate the desired fragment size. To determine the exact incubation time for a given sample type, a time course study should be performed.
      Desired Fragment Size (bp) Incubation Time (min)
      50–200 25–35
      200–1,000 15–25
      1,000–2,000 10–15
      *If starting material is 100 ng or less, incubation times should be increased by 10 minutes.
    5. Add 5 μl of 0.5 M EDTA to stop the reaction.
    6. Clean up the fragmented DNA with column purification or using SPRI beads. If using SPRI beads, it is recommended to dilute the sample 1:1 with sterile water for easier handling of the sample and faster collection of the beads to the magnet.
    Bioanalyzer: Clean up the fragmented DNA prior to loading on a Bioanalyzer chip.

    End Repair: Clean up the fragmented DNA then proceed with desired DNA end repair protocol.

    Polyacrylamide Gel Analysis: Clean up the fragmented DNA prior to loading the samples on a PAGE gel.

    Long Term Storage: Clean up the fragmented DNA prior to long term storage.

    Agarose Gel Size Selection/Analysis: Samples can be loaded directly on to an agarose gel. It is not necessary to clean up the reactions prior to loading.