To ensure consistency accross samples, use a multi-channel pipette where possible. Calibrate pipettes periodically.
To avoid unnecessary freeze-thaw cycles when performing experiments of fewer than 96 samples, Illumina recommends that you aliquot smaller volumes of reagents normally stored frozen after they are thawed for the first time.
Make sure that Neutralization Buffer (NT) is at room temperature. Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended.
Always use fresh filter tips between samples and between dispensing index primers.
Mix samples with a multi-channel pipette and centrifuge the plate when indicated. Do not vortex the plates.
Pipette carefully to avoid spillage.
Clean pipettes and change gloves between handling different adapter stocks.
Clean work surfaces thoroughly before and after the procedure.
Handling Magnetic Beads
Prior to use, allow the beads to come to room temperature.
Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogeneous.
Take care to minimize bead loss which can impact final yields.
Change the tips for each sample.
Let the mixed samples incubate at room temperature for the full duration specified in the protocol to ensure maximum recovery.
When aspirating the cleared solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and to not disturb the separated magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips.
To prevent the carryover of beads after elution, approximately 2.5 μl of supernatant are left when the eluates are removed from the bead pellet.
Always prepare fresh 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh 80% ethanol should be prepared fresh for optimal results.
Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants.
Keep the reaction plate on the magnetic stand and let it air-dry at room temperature to prevent potential bead loss due to electrostatic forces. Allow for the complete evaporation of residual ethanol, as the presence of ethanol will impact the performance of the subsequent reactions. Illumina recommends at least 15 minutes drying time, but a longer drying time may be required.
Resuspend the dried pellets using a single channel or multi-channel pipette.
When removing and discarding supernatant from the wells, use a single channel or multi-channel pipette and take care not to disturb the beads.
To maximize DNA recovery during elution, incubate the DNA/bead mix for two minutes at room temperature before placing the samples onto the magnet.