Tips and Techniques

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

Remove the plate and proceed to the next step immediately after the thermal cycler program ends.

Avoiding Cross-Contamination

• When adding or transferring samples or reagent master mixes, change tips between each sample.
• When adding index adapters with a multichannel pipette, change tips between each row or each column. If using a single channel pipette, change tips between each sample.
• Do not reuse seals from plates.

Sealing the Plate

• Before starting the following steps in the protocol, seal the 96-well plate with the adhesive seal. Use a rubber roller to cover the plate with the seal.
– Shaking steps
– Centrifuge steps
– Thermal cycling steps
• Use Microseal 'B' adhesive seals, or equivalent, for thermal cycling or short-term storage. Microseal 'B' seals are effective at -40°C to 110°C and suitable for skirted or semiskirted PCR plates.

Handling Beads

• Store BLT-LR and EBLTL upright in the refrigerator so that the beads are always submerged in the buffer.
• Vortex stock reagent tubes thoroughly until the beads are resuspended. To avoid resettling the beads, centrifugation before pipetting is not recommended.
• If Illumina Purification Beads are adhered to the side or top of a 96-well plate, centrifuge at 280 × g for 3 seconds, and then pipette to resuspend.
• When washing beads: 
– Use the appropriate magnetic stand for the plate.
– Keep the plate on the magnetic stand until the instructions specify to remove it.
– Pipette slowly to minimize foaming.
– Do not agitate the plate while it is on the magnetic stand.
– Do not disturb the bead pellet.
– If beads are aspirated into pipette tips, dispense back into the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).
– If liquid becomes adhered to the side or top of the tube or well, seal and centrifuge at 280 × g for 3 seconds to collect contents.

Handling Tagmentation Wash Buffer 2 (TWB2)

• Dispense Tagmentation Wash Buffer 2 (TWB2) directly onto the beads.
• Pipette slowly to minimize foaming.

Handling Stop Tagment Buffer (ST2)

• Pipette slowly to minimize foaming.