Hybridization and X stain Infinium Assay Troubleshooting Guide

The table below lists the most common issues observed during the Hybridization and X-stain steps in the Infinium Assay workflow, the probable cause, and ways to resolve the issue.

Symptom	Probable Cause	Resolution
There was not enough reagent to dispense to all the BeadChips.	Reagents stuck on the lid or on the side of the tubes, after thawing.	Gently invert tubes several times to mix and make sure to centrifuge the reagent tubes at 280 × g after thawing.
Programmable pipettor may be set incorrectly.	Check and correct settings of programmable pipettor.
Manual pipettor not dispensing the correct volume.	Check pipette calibration.A gravimetric test using water is a quick and easy way to check for accurate pipette dispensing volumes. Recalibrate pipettes yearly.
Cap mat deformed or melted while heat-denaturing DNA samples.	Foil heat sealer should have been used.	Use the foil heat sealer for all temperatures ≥ 45°C.If samples have been ruined. Repeat the experiment.
A small amount of precipitate was present in the hybridization solution.	A small amount of precipitate is normal and does not affect data quality.	Continue processing the BeadChips.
BeadChips are still wet on the underside after 55 minutes in the vacuum desiccator.	BeadChips must dry for a longer period.	Dry BeadChips longer under vacuum desiccator. Lab temperature and humidity affect drying time.
XC4 may be old.	Replace with fresh XC4. XC4 is reusable only up to six times during a two-week period.
An old bottle of Ethanol may have absorbed atmospheric water.	Replace with a fresh bottle of Ethanol.
After coating the BeadChips with XC4, some uncoated areas remain.	During the coating process, a bubble formed between the BeadChips and prevented the XC4 solution from reaching the surface.	Briefly place the staining rack with BeadChips back into the wash dish containing XC4. Gently move BeadChips back and forth while moving up and down to break the surface of the solution.The back and forth movement is especially important when processing 16 or 24 BeadChips.
During X-stain, the liquid in the FlowThrough Chamber dropped below the bottom edge of the glass back plate reservoir.	Glass back plates may not have been completely clean, causing capillary gap failure.	Clean glass back plates and ensure that there are no scratches and plates are not chipped.
Incorrect spacer was used to assemble the Flow Through Chamber.	Ensure that the correct spacer is used to assemble the Flow Through Chamber.
The Flow-Through Chambers have not been securely assembled.	Attach the metal clamps to the Flow-Through Chambers as described in the Reference Guide for the assay.
Difficulty in removing the IntelliHyb seal, leaving some bits of adhesive in the array.	BeadChip may have dried up due to one of the following:	Carefully remove all traces of seal adhesive. Any trace of the adhesive will impede the reagent flow.
Not enough humidifying buffer (PB2) in the chamber well.	Ensure that the top and bottom wells of each BeadChip sub-chamber are filled with correct volume of PB2.
Incorrect Hybridization Oven temperature.	Check that the Hybridization Oven temperature is correct.
Loosely screwed Hybridization chambers clamps.	Ensure that the four clamps of the Hybridization chambers are tightly screwed. Improperly sealed chamber will result to evaporation of the humidifying buffer.
Hybridization chamber gaskets are brittle or worn-out.	Check that the gaskets are in good condition.
Unusual reagent flow patterns in the BeadChip images.	Dirty glass backplates, possibly debris trapped between the glass backplates and BeadChips, or chemical deposits on the glass backplates which caused a disturbance in the reagent flow.	Clean the glass backplates thoroughly before and after each use.X-stain reagents contain components such as blocking proteins, enzymes and antibodies that can build up on glass backplates over time. Residue build-up can impede the flow of reagents and affect staining and subsequent performance of the BeadChips.

For further information, refer to the Infinium Assay Lab Setup and Best Practices.

For any feedback or questions regarding this article (Illumina Knowledge Article #5025), contact Illumina Technical Support [e-mail].