If you are running technical replicates (the eluted DNA from the same sample and running the same assay target), you may notice an alarm with the symbol “!” in the results section of your amplification plot. See image below:
The "!" alarm will signify a greater than 1 difference in Ct (cycle threshold) values for the technical replicates.
"!" Alarm Displayed
A greater than 1 difference in Ct value between technical replicates suggests inconsistency or variability in the amplification of the target sequence. It may indicate issues such as pipetting errors, variations in reaction conditions, or uneven distribution of template DNA.
Potential Actions to Take:
- Repeat the Experiment: To minimize variability, perform the qPCR experiment again with careful attention to pipetting techniques and reaction setup.
- Verify there is no Inhibition: Consider completing a 1/10 dilution with the eluted DNA and repeat the experiment
- Check for Contamination: Verify that there is no contamination in reagents or samples that could affect amplification efficiency.