The table below lists some of the most common issues observed during the precipitation and re-suspension steps in an Infinium assay workflow, the probable cause and methods to resolve the issue.
Infinium Assay: Precipitation and Re-suspension Troubleshooting Guide
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Symptom
Probable Cause
Resolution
No blue pellet observed in wells after 20-minute centrifugation.
Original DNA sample maybe degraded or the DNA input is low.
If pellets do not appear, the original DNA samples maybe degraded or DNA input is low. Repeat the Amplify DNA (Make) step of the assay protocol used.
Precipitation reaction solution was not mixed thoroughly before centrifugation.
If pellets appear, the solutions were not mixed before centrifugation. Invert the plate several times and centrifuge again.
Either PM1 or 2-propanol was not added.
Add missing reagent to wells. Inspect wells for complete mixing before the 20-minute centrifugation.
Blue color observed on absorbent pad after precipitation supernatant was decanted.
Precipitation reaction solution was not thoroughly mixed before centrifugation.
The samples are lost. Repeat the Amplify DNA (Make) step of the assay protocol used.
The plate was centrifuged at less than 3000 × g, or for less than the recommended time.
Check the centrifuge program to ensure that the correct speed was selected.
Supernatant was not removed immediately after centrifugation.
Decant supernatant immediately after centrifugation.
Blue pellet did not dissolve back into solution after vortexing.
An air bubble formed at the bottom of the well that prevented the pellet from mixing with RA1.
Pulse centrifuge plate to 280 × g to remove air bubble, then re-vortex plate at 1800 rpm for 1 minute.
Vortex speed is not fast enough.
Check the vortex speed setting, as the speed setting may drift over time. Recalibrate if necessary. Re-vortex plate at 1800 rpm for 1 minute.
The reaction plate did not incubate for long enough.
Incubate the plate for an additional 30 minutes. Make sure that the cover mat is properly seated to prevent evaporation.
Non-blue pellets but pellets are of normal size.
Blue dye may have settled in the tube.
Ensure that PM1 is well mixed by inverting the tube prior to use.
PM1 does not contain a high amount of blue dye but precipitation salts are at a normal concentration.
A slight lot to lot variation in the amount of blue dye present in PM1. Lack of color should not affect the function of the precipitation salts. The blue dye component in PM1 aids in the visual inspection of the pellets and does not have any impact on the precipitation chemistry or downstream data quality.
Proceed if the pellet is of normal size. Pellet size is a better indicator of subsequent assay performance. If the sample is degraded or of poor quality or if the DNA input is low, you may get smaller or no pellet.
For any feedback or questions regarding this article (Illumina Knowledge Article #3842), contact Illumina Technical Support techsupport@illumina.com.
