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  • Fragmentation of GC-rich Genomic DNA with NEBNext dsDNA Fragmentase (M0348)

    Introduction

    Fragmentation of genomic DNA is required during next generation sequencing workflows as well as for creating DNA inserts for expression libraries. NEBNext dsDNA Fragmentase has been optimized to fragment DNA into size ranges compatible with Roche 454, AB SOLiD, and Illumina GAII sequencing workflows. 

    DNA fragmentation by physical or enzyme based methods is affected by %GC content of genomic DNA. This application note describes the use of NEBNext dsDNA Fragmentase to efficiently fragment GC-rich genomic DNA (> 60% GC). In general, longer incubation times are required to generate 100-800 bp fragments from GC-rich genomic DNA using NEBNext dsDNA Fragmentase.

    Protocol

    1. Vortex DNA sample
    2. Set up the digestion reaction at room temperature using the following guidelines (note that dsDNA Fragmentase should not be added at this point): 

      Table I. dsDNA Fragmentase reaction conditions for GC-rich genomic DNA 
      Reaction Components Starting DNA Amount
      GC-rich gDNA (µg) 1 2 3 4 5
      10X Fragmentase
      Reaction Buffer (µl)
      2 4 6 8 10
      100X BSA (µl) 0.2 0.4 0.6 0.8 1
      dsDNA Fragmentase (µl) 2 4 6 8 10
      sterile dH2O variable variable variable variable variable
      Final Volume (µl) 20 40 60 80 100
    3. Vortex thoroughly
    4. Incubate samples on ice for 5 minutes.
    5. Add NEBNext dsDNA Fragmentase according to Table I.
    6. Vortex thoroughly
    7. Incubate at 37°C according to the recommended times below to generate the desired DNA fragment size. 

      Table II. NEBNext dsDNA Fragmentase reaction times for GC-rich gDNA
      Desired Fragments Size (bp) Incubation Time (min)
      600-800 40
      300-600 50
      100-300 60
    8. Stop the reaction by adding 5 µl of 0.5 M EDTA
    9. Purify DNA Sample on one column and elute in 35 µl of sterile dH2O or elution buffer
    10. End Repair. If DNA fragments are to be used for the Illumina Gemone Analyzer DNA sample preparation or for TA cloning of library screening, add 1 µl of E. coli DNA ligase for Fragmentase (NEB #M0349) to the end repair reaction with > 1 µg DNA or 0.5 µl of E. coli DNA ligase for Fragmentase to the end repair reaction with < 1 µg DNA.