I used Taqman based fluorescent materials with Cy5 primer & probe in Real time PCR. In test experiments, I found some late peak in NTC well. What do I fail to notice in this test?
Please let me know that the cause to me.
are the NTC you are referring to the small red peaks on the right? It might be simply some small cross-contamination or non-specific amplification. Even an NTC can give a signal if amplified for enough cycles. Perhaps 40 cycles are too much and you should set a cycle-threshold at 35 cycles. In the Life's Viia7 machine I was using, the protocol was to run for 45 cycles but then call negative everything that gave a signal after 40 cycles...
are the NTC you are referring to the small red peaks on the right? It might be simply some small cross-contamination or non-specific amplification. Even an NTC can give a signal if amplified for enough cycles. Perhaps 40 cycles are too much and you should set a cycle-threshold at 35 cycles. In the Life's Viia7 machine I was using, the protocol was to run for 45 cycles but then call negative everything that gave a signal after 40 cycles...
I would not worry
They are coming up > 35 cycles and are separated by >> 10 cycles from your actual specific signal; In other words your S:N is very high
To put this in contact, if you were to run this on an agarose gel you would get a strong specific PCR band for your real sample and nothing detectable in your NTC control
Yes ideally you would want nothing but when the final is manifest this late and you your actual signal is so much stronger and earlier it will have ZERO effect on your analysis
I agree with all the above answers!
Since qPCR is so sensitive, you will almost always detect background signal. You should assign this "level" of signal as background and that is where you consider samples that are negative to show up in your amplification graphs. I run a CFX98 and use several probe-based labels as well as SYBR, and I always see some sort of amplification after 35+ cycles.
I recommend running NTC in addition to either positive controls or standards with every qPCR run, and then keep track of where the controls always amplify and then you'll keep that for your notes. Then if you see something start to amplify earlier in your NTC, then you might consider that setup contaminated and repeat qPCR with new reagents. Keep track of all the CTs and you'll start to recognize what is "normal" and what isn't. Your amplification curves look great!
This may be your background noise since the Ct is far away than your sample value. Check your reagents for cross contamination and use fresh Nuclease free water. Keep NTC control for every Real Time PCR run for better understanding your run cycle.
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