> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/microarray/general/microarray-general-troubleshooting-list/000005025.md).
# Infinium Assay Hybridization and XStain Troubleshooting Guide
The Hybridization and XStain steps are critical stages in the Infinium Assay workflow, which directly impact assay performance and data quality. Variability or errors at these stages can lead to suboptimal signal intensities, reduced reproducibility, or assay failure. As such, it is important to identify and address issues promptly to ensure consistent and reliable results.
The table below summarizes the most frequently observed issues during the Hybridization and XStain steps, along with the probable causes and recommended troubleshooting actions.
**Table 1.** Summary of commonly observed errors, associated causes, and recommended resolutions.
| | | |
| ----------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------- |
| **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 the 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 contaminated, 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. Once reconstituted, XC4 is reusable only up to six times during a two-week period.\* | |
| \* An old bottle of Ethanol may have absorbed atmospheric water. | | |
* Something other than 100% ethanol was used to reconstitute XC4.\
\| Replace XC4 with a properly reconstituted fresh bottle using 100% 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 XStain, the liquid in the Flow-Through 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 or chips. |\
\| 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 or more of the following causes. | 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 the 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 back plates, possibly debris trapped between the glass back plates and BeadChips, or chemical deposits on the glass back plates which caused a disturbance in the reagent flow. | Clean the glass back plates thoroughly before and after each use.XStain reagents contain components such as blocking proteins, enzymes and antibodies that can build up on glass back plates over time. Residue build-up can impede the flow of reagents and affect staining and subsequent performance of the BeadChips. |
\*Further information on XC4 usage and storage on Illumina Knowledge [Recommendations for Infinium Array XC4 reagent use and storage.](/microarray/general/microarray-general-reference_material-list/000001517.md)
For further information, refer to the [Infinium Assay Lab Setup and Best Practices](https://support.illumina.com/downloads/infinium-assay-lab-setup-and-procedures-11322460.html).
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #5025), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001517%20-%20Microarray%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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