Until recently I used an ABI 7500 thermo cycler for qPCR and iTaq SYBR Green premix. With this, I always got nice multicomponent plots with a smooth SYBR Green fluorescence signal and a constant ROX signal.
Now, I have to use a different instrument, ABI StepOnePLUS, and a different SYBR Green Mix, Maxima SYBR Green, and I also tested SYBRUp but the fluorescence signals look very strange to me. Also the ROX signal is not constant anymore though I am using the same primers and concentrations as before. (See 2 files attached)
Who has experience with qPCR and has an idea about the problem I face?
I repeated the experiment and got similar results though I spent attention to completely thaw all samples and chemicals, to vigorously mix everything, to tightly seal the plate, and to follow the cycling conditions recommended in the user guides of the respective SYBR Green mix.
Is it possible that the plates I use are not appropriate? Does the seal also influence the fluorescence signal?
I will be very grateful for every advice you can give me!
Thanks in advance
This must be a problem with the instrument. It obviousely skips the measurement of each 2nd cycle during the amplification protocol. I have no idea for the reason, I would rather suspect an electronical than a mechanical problem. I would consult the technical service from ABI.
Dear Jochen,
thanks for your answer! Skipping every second cycle would only explain the alternating multicomponent plot, woudn't it? The other plot doesn't show the wild staggering but doesn't show a smooth curve either.
By now, I run an additional qPCR on a different instrument but with the new SYBR green and the multicomponent plot looks weird again (see attachment). This time, the ROX signal is much higher than the SYBR signal. This is another phenomenon now that I've never seen before.
For me this plot looks ok. It is not relevant if the values for ROX are higher than those for SYBR Green. It could be that the gain settings for these channels are different.
The only thing that I find strange in this plot is that the x-axis is labelled "Cycle" but the curves show the amplification and the dissociation curve data. The dissociation curve is not generated by cycling but by a continuous linear increase of the temperature, so the more correct labelling of the x-axis would be "Time" (given in seconds or so). But this might be mistake of the programmers (I don't know this software) of this multicomponent plot function.
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