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Real-Time PCR


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#1 heidimb2000

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Posted 04 October 2004 - 05:47 PM

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!

#2 turnerm

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Posted 06 October 2004 - 01:48 PM

Is your amplification plot scaled correctly? It sounds like you may just need to adjust the scaling on the graph/output.

Mary




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