Originally Posted by katienana
I want to design primers for a particular strain of bacteria, I want it to be specific only for that particular strain of bacteria(xanthomonas) and not other closely related bacteria. I have seen BLAST ,but I dont know how to ask it to design primers that wont amplify other bacteria. How can I search for that unique seqence? Help please! Thanks. Am new to it all.
I'd suggest looking for a gene that specific to your organism first then make your primers within the gene. I haven't had luck using blast to create the primers, you'll need to do it manually most likely. I pick the gene, get the sequence from Pubmed nucleotide fasta, blast that to test if it shows up in any other organism.
Once you have the unique to organism gene, use Amplify (or similar software) and manually select primer sequences within that gene to make the product the size you want. Make sure the primers don't have any dimers, and have a good Tm range for your experiment.
Then take those primers and put them in primer blast to test if they will amplify anything else.