get the sequences of your gene of interest from many different bacterial phylums, and same genera and all related species > do a sequence alignment > find a region which is unique for your species > design primer from that region > do an in silico check > do experiment with designed primer > do validation with PCR fragment > if did not worked > repeat/redesign your primer.

get the sequences of your gene of interest from many different bacterial phylums, and same genera and all related species > do a sequence alignment > find a region which is unique for your species > design primer from that region > do an in silico check > do experiment with designed primer > do validation with PCR fragment > if did not worked > repeat/redesign your primer.

Hi, in addition to the previous reply i would recommend you to use the Primer designing tool from NCBI. Here is the link : https://www.ncbi.nlm.nih.gov/tools/primer-blast/

It will help you to find quickly couple of primers that you can test further in silico with other tools such as FastPCR software.

Good luck

B.

You don't need genes, in fact if you are designing primers flanking genes you won't have any resolution between closely related individuals (because genes that code are conserved and you need variability). You need to design primers for microsatellites. If there is genome information in genbank, you can use msat commander (software) to search for microsatellite regions.

Here is a paper that I wrote to design microsatellit markers for a cactus species with no genome data available.

Article Microsatellite loci development for rare Colorado Sclerocact...

And another for designing primers when you have genome information available (in GenBank)

https://www.biorxiv.org/content/10.1101/332320v1.full

I hope that helps.

https://www.youtube.com/watch?v=hyrpRaYjqDE

http://primer3.ut.ee/

thank you all for responding