I am using primer & Probe concentration 10 uM and using 0.5 uL in 25 uL reaction PCR setup. I have used so many different water but each water coming positive. Its might be primer dimer but fluorescence value is also high. With Primer Dimer its possible high fluorescence value? However, my IC is coming good and no signal in water. Please suggest if any idea.
water should be negative control. it may be contaminated from posaitive control or the reaction mix.
In real time PCR, ct value is much more important than total fluorescence signal. In this graph, the signal from water sample start after 33 cycle and does not qualify typical exponential growth curve. These late signal would not affect the signal from your test sample. It appears fine to me. And yes, primer dimer can produce this signal You can check it by running the qPCR product in agarose gel.
Hi,
how did you optimize the protocol? I would think about running a gradient. In addition you could check on a gel if there is a product or only Probe hydrolysis or so is the reason. Your IC is also quite early, perhaps you see also a kind of fluorescence crosstalk? You cold try to run the assays individually behapt this help. Which chemistry are you using?
Sven
Ravi Dubey Thank you for your reply. It might not affect the regular samples, but water should not be positive right? here in my assay, whatever samples i have run its showing positive signal at same time for each sample. I thought it might primer dimer, but dimer should not cross the certain amount of fluorescence value as much i know. So, i am confused whats the problem. ?
Sven Reister Thank you for your reply. I am using taqman chemistry, and IC which i am using is MS2 strain. So IC CT value is always between 15-20. My problem is each and every samples CT value started at same time even water also. I have changed all the reagents, and autoclaves water and tips, still is showing the same.
In fact this is hard to say here. I would check if a gradient PCR improves your results. This could help in case it is a specificity problem. Apart from this some sequences tend to show hydroliysis more often. Is this an assays you are running sometime already and the problem appeared recently only?
Sven
I agree with most of colleague. you should firstly clean the PCR area. check the quality of DNA then try to change the stocks. after that if we see the same problem still present or not
Maulik Rachh Raw data of this experiment will explain the cause correctly. This graph is showing the normalized fluorescence signal with passive dye. Ideally, there should not be any fluorescence signal in NTC sample but if it is, that not always because of DNA contamination. DNA amplification graph should follow typical exponential pattern. For other signals, You should analyse raw data of fluorescence (SBGR+ROX).
Sven Reister Recently i found these problem form last 2 months. Earlier i never got the positive signal in water.
1- check you primer dimerism potential.
2- use sterile distilled water and be sure to autoclave it well.
3- try to change the environment you are carrying in your PCR mix
4- using the UV for decontamination is crucial step
5-make sure to autoclave the PCR strip and never open it before preparing your mix
Good luck
Hi Maulik,
In my opinion, DNA contamination in negative control (water) is not the problem here. This kind of curve sometime occurs in SYBR-green chemistry, where you will see amplification towards the end (mostly near 40 cycle) of realtime-qPCR in the negative controls. It happens because of primer-dimer formation. But since you said you are using taqman chemistry, it should not happen.
I believe that the problem is optimization of protocol. Like Mr. Sven Reister suggested, try gradient PCR. Also, check the instrument protocol and the protocol provided my the probe manufacturer. Sometime, one degree of temperature difference or slight change in time or cycle greatly improves the result.
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