Primer-BLAST... - (Jul/08/2010 )

Hi there,

I'm trying to design primers using NCBI Primer-BLAST. I'm amplyfing from mouse genomic DNA and want to check if my primers are specific, so under "Primer Pair Specificity Checking Parameters" I selected mus musculus as the organism and "Genome (reference assembly form selected organisms)" as the database to check against. Now, the website says that mouse is definitely one of the "selected organisms" included in that database. Nevertheless, I always get the following error:

Primer specificity cannot be correctly determined as sequences from the specified organism (Mus musculus) are not present in selected database: primerdb/genome_selected_species.

Has anybody experienced this? Any tips for a fix/workaround? It works if I choose to check specificity on the genomes of all organisms, but the search takes much longer then.

Cheers,
Matt

-mjlm3-

I've now been trying my luck with Primer-BLAST for hours and am starting to doubt that it works at all. I'm trying to check the specificity of existing primers, and some sort of error comes up every time. For example, it declares that one of my primers cannot be found in the template although it absolutely definitely is in there. Does anybody has experience with this tool? Am I missing some counter-intuitive settings? Is there a tutorial explaining the use of primer-blast or something like that?

-mjlm3-

I would only select organism as Mus musculus and leave the default Refseq database.

I usualy just leave most of the settings on default and it works well. But I use Primer3 as my primary primer designing tool.
As for the primer not in sequence, be sure you copied the sequences without any whitespace on the beginning or end, aplication may not recognise the correct sequence.

Just for test, what are your primer and template sequences?

-Trof-

Another error I often get is a blank page saying "Empty Result" at the top...

@Trof: Does it make sense to use the default Refseq_RNA database if you amplify from genomic DNA? After all, I also want to avoid unspecific amplification of non-transcribed regions of the genome.

-mjlm3-

mjlm3 on Jul 9 2010, 03:48 PM said:

@Trof: Does it make sense to use the default Refseq_RNA database if you amplify from genomic DNA? After all, I also want to avoid unspecific amplification of non-transcribed regions of the genome.

You're right it doesn't. I didn't noticed it's a RNA reference database not DNA. My mistake.

Anyway, I tried now to check some mouse primers with PrimerBlast, selecting "Genome (reference assembly form selected organisms)" and "Mus musculus" as organism and it showed me results without any problem.

-Trof-