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  • Prebind MBD2-Fc Protein to Magnetic Beads (E2612)

    Before proceeding, you need to determine two values, the amount of total DNA that is required for subsequent experiments (e.g., NGS library construction) and the volume of prepared beads required for enriching microbial DNA from your sample. The first value, input DNA, is determined by your desired output and the ratio of host DNA to microbial DNA in your sample (which will vary depending on DNA source). This will then determine the second value, which is the ratio of input DNA to enrichment beads. For example,

    • If you need 100 ng of output DNA (enriched DNA) for library construction, and your sample has a 10:1 ratio of human DNA to microbial DNA, you will need 1 μg of total input DNA.

    • For every 6.25 ng of input DNA you will need 1 μl of MBD2-Fc-bound magnetic beads for enrichment; therefore, if your total input DNA is 1 μg, you will need 160 μl of MBD2-Fc-bound magnetic beads to isolate 100 ng of enriched DNA.

    • The amount of MBD2-Fc-bound magnetic beads required (Y) can be calculated using the following equation:

      Y  =  amount of MBD2-Fc- bound magnetic beads (μl)  =  Input DNA (ng) / 6.25 ng/μl
    The following protocol will yield 160 μl MBD2-Fc-bound magnetic beads, enough for a 1 μg input sample. For other input sample sizes, scale accordingly.

    1. Resuspend NEBNext Protein A Magnetic Beads by gently pipetting the slurry up and down until the suspension is homogeneous. Alternatively, rotate the tube gently for 15 minutes at 4°C. Do not vortex.

    2. Prepare 1X Bind/wash Buffer on ice by diluting 1 part NEBNext Bind/wash Buffer (5X) with 4 parts DNase-free water. One individual reaction, from start to finish, will require 4 mls of 1X Bind/wash Buffer.

    3. In one tube, add 16 μl of MBD2-Fc protein and 160 μl of Protein A Magnetic Beads. For input sizes other than 1 μg, add (0.1 x Y) μl of MBD2-Fc protein and (Y) μl of Protein A magnetic beads. Mix by pipetting up and down a few times.

    4. Mix the bead-protein mixture by placing the tube in a rotating mixer for 10 minutes at room temperature.

    5. Briefly spin the tube and place 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 with a pipette without disturbing the beads.

    7. Add 1 ml of 1X Bind/wash Buffer (kept on ice) to the tube to wash the beads. Pipet up and down a few times to mix.

    8. Mix the beads on a rotating mixer for 3 minutes at room temperature.

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

    10. Carefully remove the supernatant with a pipette without disturbing the beads.

    11. Repeat steps 7–10.

    12. Remove the tube from the rack and add 160 μl of 1X Bind/wash Buffer (kept on ice) to resuspend the beads. For input sizes other than 1 μg, add (Y) μl of 1X Bind/wash Buffer to resuspend the beads. Mix by pipetting up and down a few times. 

    13. The MBD2-Fc-bound magnetic beads are stable for up to 7 days at 4°C.