Should I use the Basic Local Alignment Search Tool (BLAST) at NCBI to check my designed primers?
While BLASTing primers is not necessary, it is a recommended step that can provide a higher confidence in the designed primers and assure that the expected amplicon in PCR is amplified from the intended target of interest. A more intensive comparison of the primer’s sequence against the GenBank database using BLAST can be very informative and avoid mispriming to unintended targets. A Primer-Blast tool is available at NCBI at http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ,where the designed primers can be directly entered and checked for unintended products. Also, primers can be checked individually against NCBI’s nucleotide database using the BLAST tool available at http://blast.ncbi.nlm.nih.gov/Blast.cgi .
