I have recently designed a set of primers to amplify a fragment of a gene present in an cDNA template. I have performed a blast search on my primers and the results have shown a hit with my gene.
However, after conducing the PCR and cloning my fragment into a vector, i sent it off for sequencing. The results were surprising. Sequence analysis showed that my fragment was from a completely unrelated gene.
I was wondering, bearing in mind my designed primers are specific for my fragment of interest, why i amplified a non-specific fragment.
Any suggestions would help.