Optional Protocol for Eluting Captured Host DNA - NEBNext Microbiome DNA Enrichment Kit (E2612)

This step elutes the captured host DNA from the MBD2-Fc-bound magnetic beads. This DNA is now free of prokaryotic, viral and microbial DNA.
  1. While the tube with the beads (section "Collect Enriched Microbial DNA", Step 2) is still in the magnetic rack, add 1 ml of 1X Bind/wash Buffer (kept on ice) to wash the beads.

  2. Carefully remove the wash buffer with a pipette without disturbing the beads.

  3. Add 150 μl of 1X TE and 15 μl of Proteinase K to the sample pellet. Mix beads by gentle vortexing (1,200 rpm) or by flicking the tube. (Note: The pellet may be difficult to resuspend initially due to the high concentration of genomic DNA bound to the beads.) Incubate the slurry in a heat block or thermomixer set at 65°C for 20 minutes, with occasional mixing (3–5 times).

  4. Briefly centrifuge the sample at 13,000 rpm in a microcentrifuge.

  5. Place the tube on the magnetic rack for 2–5 minutes until the beads have collected to the wall of the tube and the solution is clear.

  6. Carefully remove the supernatant to a fresh microcentrifuge tube.

  7. This eluted sample contains the methylated host DNA. Store this sample at –20°C, or proceed directly to purification via Ampure bead clean-up or ethanol precipitation .