3 years ago
Hello there! My multiplex PCR has decreased in sensitivity even though I have not changed any of the parameters. The materials are in good condition and the PCR machine is well calibrated. I have tested multiple samples but one of my targets has decreased in sensitivity.
Experiment: PCR Multiplex PCR - Mammalian DNA
One possible explanation is that the presence of multiple primers has induced competition among them regarding their binding potential to ssDNA. In order to circumvent this, you should try to reduce the concentration of primers that result in brighter bands. Reduce the concentration of reverse primers and try to increase the concentration of primers that correspond to weak bands.