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Plateau height qPCR

qPCR plateau rotorgene

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3 replies to this topic

#1 Denise2910

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Posted 10 August 2012 - 12:19 PM

I am struggling with my first qPCR on a Rotorgene Q with a SensiMix SYBR No ROX Kit. While my colleages get plateaus of ~100 (mouse DNA), I only get fluorescence levels of about 35 (on raw channel). I am working with bird DNA. My amplicon is about 100 bp. My annealing temperature I optimized by a normal gradient PCR.

What is the minimum plateau level I should have on a qPCR? I did not do a standard curve yet to calculate efficiancy bcs. I was not sure if it´s worth working on that with such low fluorescence plateaus....

I already tried a reaction with a higher gain, but this didn´t change anything....

Thank you very much for your help!


On the graph you see 2 samples in triplets....Attached File  qPCR Test - Magnification.pdf   142.4KB   289 downloads

Edited by Denise2910, 10 August 2012 - 12:28 PM.


#2 leelee

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Excellent

Posted 10 August 2012 - 05:25 PM

Your curves look beautiful to me. They have the right shape and are well above background, your negative control is nice and flat.
I think you are worried over nothing.

#3 ascacioc

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Excellent

Posted 10 August 2012 - 09:19 PM

I agree with Leelee: the curves look perfect. But if you are still untrustful and think that there is a limititing reagent/component in your case versus your colleagues, there are several things you can check:
-old SensiMix kit: there are things in the reaction mix that can get degraded when the kit gets too old: dNTPs that get degraded from repeated freeze-thaw; they can get limiting after a few steps and then the plateau can be reached before the natural one; SYBR Green can also lose its fluorescence over time; the DNA polymerase in the kit can also get degraded of time, also can be limiting reagent that leads to lower plateaus
-amount of primers you add vs the template: maybe the primers are used up because of too much template and this also can lead to premature plateau

Andreea

#4 Denise2910

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Posted 13 August 2012 - 04:03 AM

Thank you very much for your fast replies! I actually talked to the company this morning and they told me to do a gain optimization. This actually solved my problem (don´t know what went wrong with the first try to set a higher gain?). My fluorescence now goes up to 90 :-)





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