Newbie - Primer-Blast confusion

[brightelf]

Hello all,

I have been tasked with finding primers for fungal organisms with sequenced genomes. So, these are the steps I followed:
1) got the accession numbers for the sequenced genome
2) put the details in Primer-Blast and for primer-pair specificity, selected
(i) genome (reference assembly from selected organism)
(ii) put in the organism for the one mentioned earlier, i.e. organism whose accession # was given in the query field

All well and good. I get a list of primers, I select one pair and now I want to check in the whole assembled genome whether this primer pair will hit the specific organism from which I had earlier "created" the primer pair. So I do a Blast search for it, but I get a message -

"resulted in an empty database " (or something similar)

That is super confusing to me. If the primer was selected from said genome, should it not have given "hits" in the same genome? If no, how will I know when I run the qPCR that it will detect the said organism in a mixed sample?

Am I missing something very basic here?

Thanks for any insights

[WhatsOEver]

Though I'm not familiar with NCBI primer-blast, it seems to be a megablast/blastn related problem. When you are running a standard nucleotide blast, megablast is selected by default, but it doesn't work (well) with short input sequences (like primers). Under "program selection", choose "Somewhat similar sequences (blastn)" and try again.

[Rutia]

Maybe you'll find this post helpful for designing primers:

http://www.genomecompiler.com/tips-for-efficient-primer-design/