I do not reach plateau in my fluorescence curve. It just keeps on going up for all of my treatment samples. I am using 45 cycles and my primer: beacon pairs are optimal. What is going wrong? I increased my primers to 500nM and my beacons to 250nM conc. The target sequence is expressed in low amounts but I don't think that should be the problem. Should I add another cycle so that I have an denature anneal and extend program? Or do I need to extend my annealing time? Please Help! I am in dire need!
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