Designing one primer pair is easy. Designing 10 that work in the same tube is hard. PP6 includes a that designs sets of primers with similar Tm values that will not cross-react with each other.
Run the "BLAST" integration. Primer Premier 6 can BLAST your primers against the NCBI non-redundant database. If your 18-mer primer matches a 16-mer sequence elsewhere in the genome, that explains the extra bands. primer premier 6
The most critical parameter in primer design is the melting temperature ($T_m$). Primer Premier 6 employs the nearest-neighbor thermodynamic algorithm, widely considered the most accurate method for predicting $T_m$. This ensures that the forward and reverse primers will anneal to the DNA template at the same temperature, creating a synchronized and efficient amplification cycle. Designing one primer pair is easy