I am trying to find where my primers (1391f/EukBr) align to S. cervisiae 18S rRNA in order to eventually trim the reference database, however it is not working…any suggestions?
What’s not working? Are you sure that your 18S rRNA gene sequence is truly full length and includes the primer region? Are you also using the reverse complement of the primer sequence?
So I was initially using NCBI and PrimerBlast to try to find where they aligned. For some reason, the forward primer would align, but the reverse wouldn’t. I decided to download UniGene and the same fasta file for S. cerevisiae that I had been using in NCBI. When I did this I was able to find both primers in the sequence. I am not sure why NCBI couldn’t find them.