Tips and Techniques

Protocol Continuity

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Follow the protocol in the order described using the specified parameters.

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Avoid extended pauses until RNA is converted into double-stranded cDNA.

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

Avoiding Cross-Contamination

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When adding or transferring samples, change tips between each sample.

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When adding adapters or primers, change tips between each well or each tube.

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Remove unused index adapter plates from the working area.

Handling Reagents and RNA

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Avoid multiple freeze-thaw cycles of input RNA.

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You can store RNA in RNase-free water or TE buffer at -85°C to -65°C for up to one year.

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If you must reuse the sample, aliquot 7.5 μl or less into separate tubes for single-use.

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Keep thawed reagents on ice until needed. Promptly return all reagents to storage after use.

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When not in use, seal plates and close lids to limit contamination.

Handling Beads

The protocol uses two types of beads: RNA Purification Beads and Agencourt AMPure XP. Each bead type has a specific technical application and cannot be substituted.

Apply the following techniques when handling beads:

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Use beads at room temperature.

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Do not use beads that have been stored < 2°C.

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Vortex beads before each use and frequently throughout the protocol to resuspend. Resuspended beads are evenly distributed and homogenous in color.

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Dispense liquid directly onto bead pellets so that beads on the side of the wells are wetted.

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When the plate is on the magnetic stand, do not agitate the plate or disturb the bead pellet.

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If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, then wait until the liquid is clear (~2 minutes).

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If beads adhere to well walls, centrifuge at 280 × g for 3 seconds, and then pipette to resuspend.

Sealing the Plate

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Use Microseal 'B' adhesive seals throughout the protocol. The seals are effective at -40°C to 110°C.

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Cover the plate with the seal, and seal with a rubber roller or wedge.

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After each use, discard seals from plates.

Plate Transfers

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When transferring volumes between plates, transfer the specified volume from each well of the first plate to the corresponding well of the second plate.

Mixing and Centrifugation

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The protocol includes the choice to mix by shaking or pipetting. Use the applicable magnetic stand as follows:

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If you choose to mix by pipette, use a Magnetic Stand-96 or DynaMag-96 Side Magnet.

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If you choose to mix by shaking, use a DynaMag-96 Side Magnet.

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At any step, centrifuge at 280 × g for 10 seconds to consolidate liquid or beads in the bottom of the well to prevent sample loss.