Primer-Blast - (Nov/13/2009 )
I have a little question.:
if you design expression-primers using primer-blast or another primerdesign software, is it necessary that the primers are separated by at least one intron on the corresponding genomic DNA? In primer-blast you can click on this function, but then you find less primers...
its not necessary but always better to do that....you can detect contamination of gDNA easily in RNA.because your mRNA will not have the introns.so if you have gDNA contamination,your pcr product size will be different
Sorry, I need a little more info on this. When you say, you are designing expression primers that means that you are only looking at the promoter of the gene of interest by hooking it up to a reporter gene. I am quite lost where the intron comes into picture. Pardon my ignorance and thanks for clarifying.
i wanted to check expression by amplification of cDNA of mRna.
But i thought that, if you click on: "primer has to span exon-exon boarders", that this would automatically exclude gDNA, cause only mRNA has exon-exon boarders. While genomic DNA always has an intron between this exons. and because of this, it wasnt necessary to click: "primers has to be separated by an intron"