When I do real time PCR, sometimes I get an amplification signal in the water sample. I use AB systems Fast SYBR Green master mix (Cat NO: 4385612). I setup the experiment along with house keeping gene, interest gene with control and test sample along with the water. I can see the amplification (Ct values) curve in the sterile water sample both for the GAPDH and Test gene. When I run the agarose gel I cannot see any bands in water sample.
There are a couple of common reasons to cause non-negative amplification of negative control (water).
1. Possible contamination of your water. Try use another fresh (unopened) water. In regular PCR practice, always keep the water vial or bottle closed after uses. Because aerosol of templates or other contaminants can easily get in and ruining your water. The best practice is to have separate mix-making hood or area, which is away from your templates.
2. SYBR-Green chemistry binds to any double-stranded DNA, including primer dimers. So, if your PCR condition is not optimized, you will see amplification even in negative control, which are most likely contributed by primer dimers. The best indication is to look at your efficiency of the qPCR reactions. If it is >100%, most likely it has primer dimer build-up in the reactions. That is why, using probe-based qPCR chemistry can give better results and higher specificity.
Good luck!
Hi, can you see a correct sigmoidal curve? There is the possibility that you have contamination of fluorochrome in the water or plate itself. Do you have the possibility to send the amplification curves of the water sample and of some other sample?
Hi,
Lucia Thank you and please find the attachment for your perusal. Plate is not contaminated because I saw the rest of the wells only with water (means no primers of either one)
How many samples do you have? If you want I can see the file of PCR to se as better as possible the curves. I use a 7500fast, but I have also the software for 7000 and ht7900. Probably is a contamination of the water
There are a couple of common reasons to cause non-negative amplification of negative control (water).
1. Possible contamination of your water. Try use another fresh (unopened) water. In regular PCR practice, always keep the water vial or bottle closed after uses. Because aerosol of templates or other contaminants can easily get in and ruining your water. The best practice is to have separate mix-making hood or area, which is away from your templates.
2. SYBR-Green chemistry binds to any double-stranded DNA, including primer dimers. So, if your PCR condition is not optimized, you will see amplification even in negative control, which are most likely contributed by primer dimers. The best indication is to look at your efficiency of the qPCR reactions. If it is >100%, most likely it has primer dimer build-up in the reactions. That is why, using probe-based qPCR chemistry can give better results and higher specificity.
Good luck!
Hi Kejun and lucia
Thank you for informative suggestion and asnwer. Because I borrowed the water from neighbour lab (Ambion) there it could b contaminated.
Hi Lucia find the attachment
Hello Ramesh,
As Kejun mentioned before, I think could be the primer dimers. Have you check your melting curves? Do you have a peak in you negative control sample? Sometimes, if there is no template available, the primers tend to form dimers, and that will give you a "fake" amplification curve and Ct value.
Hello Ramesh, I have seen the PCR and I've asked also an advice to a colleague. you do not have water contamination, you have primer dimers, which is actually quite frequent and normally not troublesome. In fact, if you look at the GAPDH you notice that the melting temp of the water samples is different from that of the 29319x samples, i.e. lower, as expected for such small DNA fragments. Such primer dimers yield Cts though they are not a problem for GAPDH because they are higher enough than those of samples (29319x). Not the same for the APP gene, as Cts of water are quite similar to that of samples (especially 293-196), a situation that must be avoided in order to get reliable quantification. One question: how do you explain that HTxxx samples have worse GAPDH but better APP, with respect to 29319x samples?
Anyhow, I would say that probably the problem could be the RNA quantification or extraction. In fact, from one hand HTxxxx samples have high Cts and bad replicates, which is typical of poor RNA extraction. On the other hand, even if 29319x samples have nice triplicates, mean GAPDH Ct values of the 6 different samples are too deviating, which indicates poor quantification.
hi Lucia,
Thank you for your valuable and informative response. ya from HT22 XXX samples RNA yield is less and that too it is dilute as I diluted 293 cells
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