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  • Fragmentation of AT-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 AT-rich genomic DNA (> 60% AT). In general, shorter incubation times are required to generate 100-800 bp fragments from AT-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 AT-rich genomic DNA 
      Reaction Components Starting DNA Amount
      AT-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 AT-rich gDNA

      Desired Fragments Size (bp) Incubation Time (min)
      600-800 10
      300-600 15
      100-300 20
    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. Proceed to End repair in appropriate library prep protocol (E6040 or E7370)
    11. 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. This is only necessary for E6040 protocol, not E7370.