Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding 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.

Sealing the Plate

• Always seal the 96-well plate before the following steps in the protocol:
– Shaking steps
– Mixing steps
– Centrifuge steps
– Thermal cycling steps
– Vortexing 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.
• Foil seals are effective at -70°C to 105°C, and suitable for skirted or semiskirted plates.
• 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).
• When multiple plates are used in a step, such as a plate 1 and a plate 2, transfer volumes from the existing plate 1 to the new plate 1. Transfer volumes from the existing plate 2 to the new plate 2.

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 maintain beads in suspension, centrifuge at 100 × g for 20 seconds.
– To pellet beads, centrifuge at 280 × g for 1 minute.

Handling Beads

• Pipette bead suspension slowly.
• When mixing, mix thoroughly.
• To remove supernatant, use a multichannel pipette and 200 µl barrier pipette tips with the plunger down. Place the tip opposite the aggregated beads and aspirate the supernatant. Dispense any aspirated beads back into the plate, and then leave the plate on magnet for 2 minutes or until the liquid is clear.
• To avoid sample loss, confirm that no beads remain in pipette tips after resuspension and mixing steps.
• When washing beads:
– Use the appropriate magnet for the plate. For more information, refer to Consumables and Equipment.
– Dispense 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.
– Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.