A "Good Primer" usually follows these criteria (default settings in Primer3):
1. Length
18-25 bp. Too short = low specificity; Too long = low efficiency.
2. Melting Temp (Tm)
55°C - 65°C. Max difference between Forward & Reverse < 2°C.
3. GC Content
40% - 60%. High GC causes secondary structures.
4. 3' End Stability
End with G or C (GC Clamp) to help polymerase binding.
Specificty is Key!
Primers must be unique in the genome. Our tool uses BLASTn to check specificity against the selected Rosa genome CDS.
High Specificity = Matches only the target gene.