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.
- 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.
- Carefully remove the wash buffer with a pipette without disturbing the beads.
- 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).
- Briefly centrifuge the sample at 13,000 rpm in a microcentrifuge.
- 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.
- Carefully remove the supernatant to a fresh microcentrifuge tube.
- 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 .