To make sure there is consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.
Minimize freeze-thaw cycles to no more than four. If you do not intend to consume the reagents in one use, dispense the reagent into aliquots after the initial thaw and refreeze the aliquots in order to avoid excessive freeze-thaw cycles. However, if you aliquot, you might not have enough reagents for the full number of reactions over multiple uses.
To minimize sample loss from manual resuspension, use the recommended plates and plate shaker to mix samples.
Handling Magnetic Beads
Prior to use, allow the beads to come to room temperature. Use a 25°C water bath as necessary.
Immediately prior to use, vortex the beads until they are well dispersed and the color appears homogeneous.
After adding the beads to your samples, mix thoroughly using a plate shaker at the recommended speed and time. For some steps where complete resuspension of the beads is essential for optimal performance, after a timed shake Illumina recommends pipetting the sample up and down 10 times to ensure complete resuspension of the sample.
For best results, incubate your bead/sample mixtures at room temperature for the entire duration indicated in the protocol. After the recommended incubation time, collect the bead/sample mixture to the bottom of the plate with a brief centrifugation.
After placing the plate on the magnetic stand, wait for the solution to clear before proceeding. Keep the plate on the magnetic stand when slowly aspirating cleared solution, taking care not to disturb the separated beads.
Always use fresh pipette tips between samples and between dispensing index primers.
Mix samples using the methods specified in the protocol.
Using aerosol-resistant pipette tips reduces the risk of nucleic acid carry-over and sample-to-sample cross-contamination. If aerosol-resistant tips are not available, ensure careful pipetting to avoid contamination.
Washing During SPB Clean-Up
Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air impacting your results.
Make sure that you remove all ethanol from the bottom of the wells as it might contain residual contaminants. Use a P20 multichannel pipette to remove residual ethanol and accelerate drying.
Allow at least 10 minutes of drying time on the magnetic stand at room temperature for complete evaporation. Residual ethanol can impact the performance of subsequent reactions.
Do not exceed the recommended drying time as overdrying samples can negatively impact sample recovery.
Freeze/thawing a Small Number of Samples
Each reagent supplied with your assay kit contains sufficient volume to process the kit specific number of samples at one time using an 8-channel pipette and either a reservoir or an 8-well strip tube. When processing smaller sample batches using a reagent reservoir, dead volume and pipetting error losses can increase. To make sure there is an accurate reagent volume for all samples, single-pipette the reagent into each well.
To store remaining reagent, Illumina recommends freezing aliquots, rather than repeatedly freezing and thawing the supplied reagent tubes.
Prevent PCR Product Contamination
Make sure that the lab is set up appropriately to reduce the risk of PCR product contamination. For more information, see the Prevent PCR Product Contamination section of the Nextera Rapid Capture Enrichment Guide.
Low-Plex Pooling Guidelines
Illumina uses a green laser to sequence G/T and a red laser to sequence A/C. At each cycle, at least one of two nucleotides for each color channel must be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure.
Follow the instructions described in the Low-Plex Pooling Guidelines section of the Nextera Rapid Capture Enrichment Guide when pooling less than six libraries, to determine which libraries are pooled pre-enrichment.