I cannot design primers on exon-exon junction - (Jan/26/2014 )
I am trying to design a primer using NCBI's Primer Blast for real time PCR. I limited my product size from 70 bp to 200 bp. I also choose "Primer must span on exon-exon junction". Since all of the exons are larger than 200 bp, I did not get any primer that match my criteria.
I am just wondering if I do not design primer on exon-exon junction for my qPCR, what other measure can be taken to rule out/ discriminate DNA contamination?
Increase minimum and maxmimum size of primers:
The above post should give you a primer pair that will span an exon. You may have to loosen the stringency of the design. I am not familiar with the tool you cite above, so not sure if can be done.
My concern would be in the reaction that you set up. You would need to lengthen the amount of time for the elongation step of the RT-PCR in order to visualize a fragment of DNA with the intron. Why not design a primer spanning the intron/exon border? Or do you know that the intron is really small (100 to 300 bp) and will show up readily?
Do you plan to run the RT-PCR products out on an agarose gel or to do a "regular" PCR reaction to test for genomic DNA contamination?