Have you ever tried to optimize primer using Ranger PCR system where the expected length of the product is 500 bp long? The reason I thought of Ranger as I tried Amplitaq, Expand, Superfi II and non of these systems worked. I re-designed the primers three times hoping to solve this problem ,but without luck :( .
keep in mind I am dealing with very polymorphic gene and the the exon located between too large introns , so there is no chance to design my primer on the adjacent exons which are maybe less ploymorphic .
the target exon also contains a lot of repetitions of the A and T and CG rich
if you have any thoughts or hints I can do to solve this issue, please don’t hesitate to put here .
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