I've been fighting with Primer Blast for a couple of days already. I'm trying to get primers for RT-PCR for 2 genes. Because of the mRNA template, I'm choosing the option "exon-exon junction spanning" BUT the Primer Blast cannot see any exons in some RefSeq templates although they ARE listed in the reference sequence.
the Claudin 7 gene has 3 transcript variants.
- I enter accession number of the transcript variant 2 -> here they are, primers specific for all 3 transcript variants, beautifully spanning exon-exon junctions in all 3 transcript variants!
- I enter accession number of the transcript variant 1 or 3 -> "Exon/exon junction cannot be found for submitted PCR template"!
The second gene has only 1 transcript variant and it does not search for primers because also "Exon/exon junction cannot be found for submitted PCR template". Although exons ARE there, I tell you!
I tried other online tools, sometimes they may help but I would really like to have the same algorythm for all genes I need (I'll need primers for more genes in near future)!
I feel solution is somewhere near only I don't see it... perhaps I should just check some option or something like that.
Thanks to anyone who could help me!
UPDATED: I've just repeated the SAME search and it worked! Now I don't understand it at all! Perhaps the tool does not work when it's overloaded with queries? Or it just is frightened by the fact I wrote a complaint here on the forum?))
Edited by Maharisha, 10 January 2014 - 09:14 AM.