I have designed a pair of primers for amplifying an truncated gene. Since I wanted to clone the PCR product in pET28a vector, I have added the restriction sites for NheI and XhoI to the forward and reverse primer sequences, respectively. The product size should be around 600bp. But after ampilfication, I just detected an single band of around 500bp. I was wondering if the primers have nonspecific binding sites in the template.
Would you please introduce me an online software which can show all possible binding sites of a designed primer in the template sequences?