I ran an assay with Cy5 probe using Bio-Rad CFX96. Why are some of the curves less intense than some of the other curves? The smaller curves are using cDNA and the larger curves are using plasmid standards. I used iScript gDNA Clear to synthesize the cDNA and the plasmid DNA is synthesized from an external company. The cycle values are very similar between replicates, so I think the data is still good, I just don't know why the shapes are so different.
These curves are background corrected. I may be that the cDNA binds a significant proportion of the SYBR Green, so that less is available to monitor the amplification. You can check this looking at the raw data and by using dilutions of the cDNA.
It may also be that the cDNA contains some component that reduces the emmission efficiency of the dye (just an idea - I don't know what this can be; maybe the pH or a residing salt, phenole, etc.). This should also be less a problem when you dilute the cDNA.
you have to repeat the experiment in order to ensure that the low RFU of the ccDNA is not random
Have you run a standard curve with differing concentrations of plasmid and cDNA? If that is the case, then perhaps there is a problem with cDNA concentration as the cDNA curves are not separated. If not, then the level of fluorescence is not that important. If your data is on the expected lines you can ignore the low Rfu.
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