Tips and Techniques

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

• 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.
• [Tubes] Open only one index adapter tube at a time to prevent misplacing caps. Remove unused index adapter tubes from the working area.
• Always seal the 96-well plate with the adhesive seal using a rubber roller to cover the plate before the following steps in the protocol:
– Shaking steps
– Thermal cycling steps
– Centrifuge steps
• Microseal 'A' adhesive film is used for thermal cycling steps to prevent evaporation.
• Microseal 'B' adhesive seals are effective at -40°C to 110°C and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' seals for thermal cycling or short-term storage.
• Microseal ‘F’ foil seals are effective at temperatures down to -70°C and are suitable for storing the 96-well plates containing the final libraries long term.

Manually mix the bead reagent container by manual inversion. Inversion mixing constitutes turning a container upside down and then returning it to its upright position.

1. Mix the bead reagent by inversion, at a rate of at least a single inversion per second.
2. Rotate bottle 90 degrees every 30s, and continually inverting for a total of 2 minutes.
3. After this time, visually inspect the inside of the container for any solid material still adhering to the walls.
4. If container inner walls are free of any residual material, bead reagent can be considered fully resuspended.
5. If reagent is still found on the inner walls, repeat the mixing step for additional 1 minute.
6. Repeat visual inspection of the container walls to ensure complete mixing. If necessary, repeat the mixing step (5).
• Prepare IDT for Illumina DNA/RNA UD Indexes as follows.
• Nextera XT is compatible with IDT for Illumina DNA/RNA Unique Dual (UD), IDT for Illumina Nextera DNA Unique Dual (UD), and Nextera DNA Combinatorial Dual (CD) Indexes.
• Pipette slowly to minimize foaming.
• Each index plate is for single use only.
• IDT for Illumina DNA/RNA UD Indexes use 10 base pair index codes that differ from Nextera XT, and Nextera XT v2 indexes, which use eight base pair index codes. Confirm that your sequencing system is configured for 10 base pair index codes.
• Centrifuge at 1000 × g for 1 minute to settle liquid away from the seal.
• [< 96 samples] Pierce the foil seal on the index adapter plate using a new pipette tip for each well for only the number of samples being processed.
• [96 samples] Align a new Eppendorf 96-well PCR plate above the index adapter plate and press down to puncture the foil seal on all 96 wells. Press down slowly to avoid tipping the volume over.
• Discard the empty Eppendorf plate used to puncture the foil seal.