Tips and Techniques

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

Recommendations

• Make sure that reagents are not expired. Using expired reagents may negatively impact performance.
• Mix reagents thoroughly prior to use.
• Do not allow more than eight freeze-thaw cycles for any reagent in this kit.
• Running a negative control is recommended to alert you to potential amplicon carryover from previous experiments. Elution Buffer (ELB) is provided to run as a no template control. There should be no amplicons produced from the negative control samples.
• Running a positive control is recommended, but may not be required. You can use RNA diluted with ELB as a positive control.
• Sequence libraries as soon as possible after pooling. Pooled libraries are stable for up to 30 days at -25°C to -15°C.

Avoiding Contamination

• Use proper laboratory practices to prevent nuclease and PCR product contamination. Nuclease and PCR product contamination can cause inaccurate and unreliable results.
• Perform library preparation in a RNase/DNase-free environment. Thoroughly decontaminate work areas with a RNAse/DNase-inhibiting solution, such as RNAseZap and DNAZap.
• Use fresh tips and fresh consumable labware between samples and dispensing reagents.
• Use aerosol-resistant tips to reduce the risk of carry‐over and sample‐to‐sample cross‐contamination.
• Due to the potential for contamination, take extreme care to make sure that well contents remain fully in the well. Do not splash contents.
• Do not use aerosol bleach sprays when performing library preparation. Trace bleach contamination can lead to assay failure.
• Use a unidirectional workflow when moving from pre-amplification to post-amplification environments.
• One or more no template controls (NTCs) are recommended per plate to monitor contamination.

Sealing and Unsealing the Plate

• Before starting the following steps in the protocol, seal the 96-well plate with the adhesive seal. Use a wedge or rubber roller to cover the plate with the seal.
– Shaking steps
– Vortexing steps
– Centrifuge steps
– Thermal cycling steps
• Make sure the edges and wells are completely sealed to reduce the risk of cross-contamination and evaporation.
• 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.
• Before unsealing:
– Briefly centrifuge the 96-well plate at 1000 × g for 1 minute. For bead steps, centrifuge at 500 x g for 1 minute.
– Place the plate on a flat surface before slowly removing the seal.

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 as needed at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.

Handling Beads

• Pipette bead suspension slowly to prevent splashing and bubbles.
• When mixing, mix thoroughly.
• 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.
– 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.