Dear Alisha

• if you subject your primer to a nucleotide BLAST search it will confirm specificity 
• As part of that process it will also map your primer to your gene target sequence in Genbank and by going to the bottom of BLAST result retrieval page you can down load the target sequence as a FASTA file
• You can then select 'find in this sequence' type in your primer sequence and then map your primer to the sequence of your gene 
• Blastn using your primer sequence from the following NCBI link:
• https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
• To do the same for your reverse primer and thereby ascertain the size of the resulting amplicon as well remember to 'find in this sequence' using the reverse compliment of your actual primer sequence. Many freeware programs exist to give you this sequence. My personal favourite is:
• http://biotools.nubic.northwestern.edu/OligoCalc.html

Dear Alisha

you might find the detailed SOP I made for students useful regarding the above

Hi Alisha,

I agree with Laurence – using BLASTn would be a good way to check for primer specificity. However, I would BLAST to all known sequences (genome, complete annotation, etc) for the species you are looking at. If your primer is specific enough your target locus should be at the top of the list. If there isn't a genome available for your species, then BLASTing to sequences from all closely related species on GenBank would be a reasonable alternative. In this case your primer should hit an ortholog or homolog. Assuming you have a genome with annotations, it is important to have the 3' end of your primer specific to your target locus. I've gotten away with having most of my primer identical to an off-target, but with 3-5 bp on the 3' end being specific to my target locus. Due to the short sequence of a primer you are by chance going to find similar motifs. Check out Primer3 for primer design if you have to go back to the drawing board, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/

Adam