Dear everyone,
I need to perform this real time PCR in order to obtain an estimation of total bacteria in my samples by amplifying the 16s rRNA gene of each microrganism. The problem is that my melting curve looks as you can see, very steep with sharp and matching peaks for replicates but the amplification in my samples is noisy and with chunky peaks. The samples consists of DNA coming from wastewater microorganisms. The point is, how can i improve the preparation or conditions to have more reliable data? Do you have any experience in the field of qPCR with 16s of bacteria?
Do you have a no template control on this picture? If it gives you a distinctly different curve then at least you can know it is not some sort of background. Chunky peaks and noise might be because of low target concentration I would guess.
Do you mean a negative, blank, control? Yes I had, unfortunately I see amplification in it and the melting curve shows a sharp peak. I think because the contaminating bacterias are not as many?
Yes, that is what I would think too. Also, many polymerases can have bacterial contamination (eg http://www.biomedcentral.com/1741-7007/12/87 ). You can also try to minimize the background amplification from NTC by raising the annealing temperature, optimizing primer concentrations or using less PCR cycles.
I know about the bacterial contamination of the polymerases because purified by ecoli, maybe it could be the reason. I will try maybe by increasing the temperature of a couple of degrees. thank you
Saverio Conti Hello, I appreciate if you could share how you solved the problem.
I have the same problem now. It seems for 16S, the NTC amplification normally happens. but my unknown samples don't show significant and sharp peak in melting curve!
I diluted the samples even to 1:1000 and it got (a little bit) better.
- Badges
- Science method