Perhaps, that could be a technical error. Run qPCR in quadruplicates. If the Cq values for example would be 22.5, 23, 23.1, 23.3 obviously remove 22.5 from your analysis.

It could be a problem of homogenization of the samples, it is recommended to vortex each tube (especially for suspensions that sediment quickly) before pipetting for DNA duplicate extraction.

A Ct (Cycle threshold) difference greater than 0.5 between duplicate samples in PCR genotyping can indicate variability in the amplification efficiency of the target DNA. Several factors can contribute to such differences:

To minimize Ct differences between duplicate samples, it's important to ensure consistent and precise experimental techniques. Consider the following steps:

• Use standardized and accurate pipetting techniques to ensure equal DNA template and reaction component volumes.
• Optimize DNA extraction methods to obtain high-quality and consistent DNA templates.
• Minimize the presence of PCR inhibitors or contaminants through thorough sample purification techniques.
• Maintain uniform PCR conditions, including primer concentrations, annealing temperatures, and cycling parameters, throughout the experiment.
• Validate the presence of template heterogeneity by analyzing duplicate samples using other genotyping methods or sequencing if necessary.

By implementing these measures, you can enhance the reproducibility of your PCR genotyping results and minimize Ct differences between duplicate samples.