You may have evaporation from the reaction well

Evaporation of the reaction can occur if the reaction plates are not properly sealed, resulting in:

• Outliers (mild to moderate evaporation)
• Trailing clusters (moderate evaporation)
• Samples clustering at the NTC (extreme evaporation)

Solution:

1. Check the wells on the plate that correspond to the problem calls. Evaporation most often occurs around the edges of the plate.
2. Check the optical adhesive cover seals for leaks.
3. If there are leaks, perform the assay again.
4. Use our MicroAmp® Adhesive Film Applicator to thoroughly seal the cover, and run the applicator over the edges of the seal.

Evaporation can occur if the plate is not properly sealed. If evaporation occurs, the signal from the reporter and ROX™ dyes increases due to an increase in concentration of the dyes. The degree of evaporation influences the assay results:

• Mild evaporation—the ROX™ dye can compensate for the increased signals and the assay will work correctly.
• Mild to moderate evaporation—outlier samples may be observed. Depending on the number of wells affected, the plot may show only a few outliers or it may show a trailing cluster.
• Extreme evaporation occurring early in the reaction—the PCR reaction fails and the samples cluster with the NTCs.