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ZymoBiomics™ 96 MagBead DNA Extraction

This is a protocol for Zymo’s MagBead DNA extraction kit. The official protocol is here. We highly recommend using bead tubes instead of bead plates to minimize well-to-well contamination.

Before starting

  • For all mixing steps, pipette mix or shake at max speed
  • Shaking speed will depend on sample volume and plate well depth. Use of shaker plates will require user optimization

Materials

  • ZymoBIOMICS™  96 MagBead DNA Kit (Zymo D4308)
  • 96-well deep well blocks (Zymo P1001-10)
  • 96-well PCR plates
  • Magnetic stand
  • VortexGenie 2 with 2 ml tube adaptor (or alternative)
  • BioShake iQ thermoshaker with adaptor for deep-well plate

Sample Lysis

  1. Add sample to ZR BashingBead tubes according to your sample type:

    2. Sample Type

    Sample TypeMaximum InputNotes
    Feces

    100 mg

    Soil

    200 mg

    Liquid Samples

    250 µL

    For water samples, filter using desired filter. Cut filter into small pieces and place into lysis tubes.

    Swab Collections

    250 µL

    Swabs can be cut or broken and placed directly into bead beading tube.

    Cells (suspended in DNA/RNA Shield or isotonic buffer, e.g. PBS)

    5-20 mg (wet weight)

    2 x 10^8 bacterial or 2 x 10^7 yeast cells

    Samples in DNA/RNA Shield (10% v/v Sample)

    250 µL

  2. Add 750 µL Lysis Solution to each tube.
  3. Vortex for 40 minutes at max speed on a VortexGenie 2 (max of 18 tubes per adaptor). See conditions for alternative bead beaters here.
  4. Centrifuge tubes at ≥10,000 x g for 1 minute.

Sample Purification

  1. Transfer 200 µL supernatant to a deep-well block (not provided).
  2. Add 600 µL of MagBinding Buffer to each sample.
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Note: For samples with excessive amounts of solid particulate, centrifuge at 4,000 x g for 5 minutes to reduce clogging.
  1. Vortex MagBinding Beads for at least one minute, then add 25 µL to each sample.
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Note: ZymoBIOMICS MagBinding Beads settle quickly, ensure that beads are kept in suspension while dispensing.
  1. Mix on the thermoshaker for 10 minutes at 1800 rpm, ambient temperature.
  2. Transfer the deep-well block to a magnetic stand until the beads pellet, then aspirate and discard the supernatant. Remove the deep-well block from the magnetic stand.
  3. Add 500 µL of MagBinding Buffer to each sample and mix on the thermoshaker for 1 minute at 1800 rpm, ambient temperature.
  4. Transfer the deep-well block to a magnetic stand until the beads pellet, then aspirate and discard the supernatant. Remove the deep-well block from the magnetic stand.
  5. Add 500 µL of MagWash 1 to each sample and mix on the thermoshaker for 1 minute at 1800 rpm, ambient temperature.
  6. Transfer the deep-well block to a magnetic stand until the beads pellet, then aspirate and discard the supernatant. Remove the deep-well block from the magnetic stand.
  7. Add 900 µL of MagWash 2 to each sample and mix on the thermoshaker for 1 minute at 1800 rpm, ambient temperature.
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Note: if high speed shaker plates are used, dispense 500 µL of MagWash 2.
  1. Transfer the deep-well block to a magnetic stand until the beads pellet, then aspirate and discard the supernatant. Remove the deep-well block from the magnetic stand.
  2. Repeat steps 10 and 11 twice, for a total of three washes with 900 µL of MagWash 2.
  3. Transfer the deep-well block to the thermoshaker and incubate at 55°C until beads dry, approximately 10-15 minutes. If no heating element is available, air dry for approximately 20-30 minutes.
  4. Add 50 µL of DNase/RNase Free Water to each sample and mix on the plate shaker for 10 minutes at 1800 rpm.
  5. Transfer the deep-well plate onto the magnetic stand for 2-3 minutes until beads pellet and then transfer the supernatant (containing the eluted DNA) to a clean PCR plate or tube.
  6. Store DNA at -20°C.