Best Practices

Ensuring Consistency

  • To ensure consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.
  • To avoid unnecessary freeze-thaw cycles when performing experiments of fewer than 96 samples, Illumina recommends that you aliquot smaller volumes of reagents normally stored frozen after they are thawed for the first time.

Avoid Cross-Contamination

  • When adding or transferring samples, change tips between each sample.
  • When adding adapters or primers, change tips between each row and each column.
  • Remove unused index adapter tubes from the working area.
  • Always use fresh pipette tips between samples and between dispensing index primers.
  • Mix samples with a multi-channel pipette and centrifuge the plate when indicated. Do not vortex the plates.
  • Pipette carefully to avoid spillage.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.

Sealing the Plate

  • Always seal the 96-well plate before the following steps in the protocol: Shaking steps, vortexing steps, centrifuge steps, and thermal cycling steps.
  • Apply the adhesive seal to cover the plate and seal with a rubber roller.
  • Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term storage.
  • Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

Plate Transfers

  • When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.
  • If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).

Centrifugation

  • Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.
  • To pellet beads, centrifuge at 280 × g for 1 minute.

Handling Magnetic Beads

  • Before use, allow the beads to come to room temperature.
  • Immediately before use, vortex the beads until they are well dispersed. Make sure that the color of the liquid appears homogeneous.
  • To avoid sample loss, confirm that no beads remain in pipette tips after resuspension and mixing steps.
  • Pipette bead suspension slowly.
  • When mixing, mix thoroughly.
  • Let the mixed samples incubate at room temperature for the full duration specified in the protocol to ensure maximum recovery.
  • When washing beads:
    • Use the appropriate magnet for the plate.
    • Displense liquid so that beads on the side of the wells are wetted.
    • Keep the plate on the magnet until the instructions specify to remove it. Let it air-dry at room temperature to prevent potential bead loss due to electrostatic forces.
    • Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.
  • To prevent the carryover of beads after elution, approximately 2.5 μl of supernatant are left when the eluates are removed from the bead pellet.
  • Always prepare fresh 60% or 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh 60%-80% ethanol should be prepared fresh for optimal results.
  • Be sure to remove all the ethanol from the bottom of the wells, as it can contain residual contaminants.
  • Resuspend the dried pellets using a single channel or multichannel pipette.
  • When removing and discarding supernatant from the wells, use a single channel or multichannel pipette and take care not to disturb the beads.
  • To maximize DNA recovery during elution, incubate the DNA/bead mix for 2 minutes at room temperature before placing the samples onto the magnet.