Hello everyone,
I am carrying out TaqMan probe based Real-Time PCR assay. I am able to observe signal peak for the NTC sample.
However upon agarose electrophoresis I was unable to see any band corresponding the target gene of interest. What could be the possible reason and how can I avoid it?
I conducted the experiment twice with different probe concentration and thermocycling conditions but, could observe signal for NTC during both the experiments. The conditions are as follows,
Dye - FAM, Quencher - TAMRA
Experiment 1
Mastermix (2X) - 10µL
Forward Primer (20uM) - 2µL
Reverse Primer (20uM) - 2µL
Probe (20uM) - 2µL
DNA - 4µL
95 °C - 10 min
Repeats - 40
95 °C - 15 sec
60 °C - 1 min
Experiment 2
Mastermix (2X) - 5µL
Forward Primer (10uM) - 1µL
Reverse Primer (10uM) - 1µL
Probe (5uM) - 1µL
DNA - 2µL
95 °C - 5 min
Repeats - 40
95 °C - 20 sec
60 °C - 30 sec
I have attached the experiment end report and the gel image for your reference.
Please help me with the standardisation of this assay.
Thanks & Regards
Yugendran
Can you include a graph of the signal over cycles? If the Ct value for your NTC is >35, it can be just an artifact (which is what I would suspect from your agarose gel).
Katie A Burnette Thanks for your response. Please find attached the graph of the signal over cycles for your reference. Please help me figure out, what could be the possible reason observing the signals for the NTC and NC.
If possibly it is because of artifact then, what measure may I take to rule them out during the future experiments.
Thanks
Yugendran
Stefan Hajny I have never performed melt curve analysis for TaqMan probe based qPCR assays. Could you please help with the analysis part. How am I suppose to analyse the samples?
Thanks
Yugendran
Most probably there is a contamination in any of your ingrediens (e.g. in water, polymerase, dNTPs or oligos).
It would be possible that you detect a signal via TaqMan(R) PCR without any band visible in an agarose gel (using this semiquantitative method, you would need much more copys/amplicons (compared to qRT-PCR) to see a signal with thenaked eye).
I would propose to check your components for contamination by replacing the individual compounds.
Looks to me like you just have an artifact. You should not see the initial huge drop in signal (your first graph) so I think the algorithm is setting the baseline too low.
You analyze your data using the delta delta Ct equation. I'd also include the Pfafl correction.
What was the Ct Value for your NTC? If it is >35, not a problem. You can compare the melt curve as well. If not, you can try with new set of reagents to rule out the possibility of contaminated reagents.
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