I am designing primers which can amplify the genomic DNA on NCBI. Everything goes well until I have my primers. But when I put them in Blast, this sequence does not correspond at all to the gene I'm trying to study. What is wrong? Is it because the primer comes from the genome? Or do I make a mistake in the design? Can someone tell me how to design this kind of primer?
Thank you very much
what software do you use to design primer?
I use the Primer-BLAST tool http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Try using the Insilico PCR tool at UCSC. If it works well at UCSC, your PCR should work well.
I usually trust UCSC results more than NCBI.(That's purely my opinion)