Hi, I designed the real time pcr primers to look for my gene of interest expression(transcript) and first checked my primers specificity using the primer blast although other variants associated to the genes were displayed after blasting .However, when I checked the primers specificity using the agarose gel with the respective cDNA, I saw a smear instead of a clean band. I tried to troubleshoot this because i suspected perhaps my RNA is degraded/contaminated and therefore used RNA as negative control and cDNA as positive control, luckily i did not see a band or smear with RNA. I went ahead and created a temp gradient from thermocycler and I still saw the smear. What can be a problem, and is there other best RT-pcr primers designing tool outs there that can help me to get clean band before doing sybr green real time pcr?
Question: Real time pcr/qPCR primers specificity
9 days ago by
muhindi_stephen • 0
muhindi_stephen • 0 wrote:
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