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brightelf 10-21-2015 02:05 PM

Newbie - Primer-Blast confusion
 
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? :confused:

Am I missing something very basic here?

Thanks for any insights

WhatsOEver 10-22-2015 01:55 AM

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 10-26-2015 07:00 AM

Maybe you'll find this post helpful for designing primers:
http://www.genomecompiler.com/tips-f...primer-design/


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