Hello,

I've been having issues with my cDNA PCR which used to work previously. I'm working with cDNA from velvet worms, amplifying Cox1. Earlier, I had successfully amplified Cox1 with specific parameters that I managed to standardize. However, suddenly it stopped working when I changed the cDNA samples, and even when using the previous sample, I couldn't get amplification. I tried using new reagents at the same concentrations and only got smears. I attempted a new cDNA synthesis, considering potential degradation, but again, only smears. I experimented with different concentrations of MgSO4 (1.2 mM, 1.5 mM, 1.8 mM, 2.0 mM, and 2.2 mM) and still only got smears. I tested various cDNA concentrations and got smears as well.

I'm unsure what else to try, and I would appreciate your guidance and recommendations on this issue.