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Old 10-21-2015, 02:05 PM   #1
brightelf
Junior Member
 
Location: usa

Join Date: Oct 2015
Posts: 1
Default 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?

Am I missing something very basic here?

Thanks for any insights
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Old 10-22-2015, 01:55 AM   #2
WhatsOEver
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Location: Germany

Join Date: Apr 2012
Posts: 215
Default

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.
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Old 10-26-2015, 07:00 AM   #3
Rutia
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Location: Israel

Join Date: Aug 2015
Posts: 12
Default

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