Primer desing - (Nov/27/2009 )
I'm just beginning the real time pcr and have some questions about the primer design. i was told that primers must be designed to span the intron to eliminate the gDNA contamination. but i'm a little confused about whether designing one of the primers spans the intron(exon-exon junction) or the product of the primers spans the intron. which one is better?
So please help me solve the question
the product should span the intron. Basically, if u hv genomic DNA contam, such a primer set will give u a bigger amplification . whereas, if there's no contam, u will see a shorter amplicon........that means there's no intron, or in other words, no gen. DNA contam....pure RNA
I think the best way is to design one of the primers across an intron exon border in that way that the 3' end of the primer is located in the intron, so you will get no amplification of gDNA. Also keep in mind that there are pseudogenes present lacking introns but are transcribed to RNA