I am attempting to incorporate two restriction enzyme sites into a plasmid vector (p15A origin PREGFP) using super primer PCR. My desired insert length is 80 bp, containing one restriction sites followed by a nonspecific sequence and followed by another restriction site. Despite several cloning attempts, sequencing of the extracted plasmid from the ligated colony showed no insertion.

Here's what I tried:

Another issue is the low concentration after super primer PCR, around 50 ng/µL. I tried changing cycle numbers and gradient PCR, but the concentration value did not improve.

I would appreciate your suggestions to resolve these issues.