The 16s rRNA gene is quantified in some surface water samples. But there was always amplification in the no template controls (which contain nuclease free water instead of DNA).
The problem is not the water. I used fresh one.
But I used Colorless 2x GoTaq qPCR master mix produced by Promega. Can this master mix contain the target gene, 16s, which is highly conserved?
Is the quantitation for unknown samples totally invalid? or is there any method to consider this NTC amplification as an overestimation factor?
Thanks
Why are you thinking about such a complicated situation. Bands in negative control simply tells about the sterile condition of your PCR assay. The tips, pipettes, working bench, PCR tubes/plate they might be contaminated. Plus how much you have breathed into and during the PCR assay.
Negative control gives information about what did go wrong and what what to do to prevent it.
Its logical if the PCR assay is contaminated, the results are not valid and no concrete points can be concluded.
Abhijeet Singh Thank you for your reply. I agree with you. But I'm quantifying other genes in parallel and they are working properly. The only thing I can think of is the master mix or the primers that make NTC amplification in qpcr targeting 16s. I thought for 16s rRNA gene the NTC amplification is quite acceptable as it is highly conserved and that's why I was looking for how to consider this in my quantification analysis.
Hi Termeh,
I would see this in a different persepctive. I is correct, that for a valid assay NTCs are ideally negative. However, this is not always possible. For 16S you can use the data as long as the level of background is marginal e.g. Cq 36 or so) and you samples give high Cqs. You will loose a bit of accuracy. However, in case you hav elow inputs some mixes are developed to be free/ very low in bacterial backgorund. In case you need such asolution, just let me know as I have developed such mixes.
Sven
Dear Termeh Hesam Mahmoudinejad you are right. Besides possible contamination of every step in your experiments (plastics, working environment, etc.) there is a chance that source of contamination is PCR master mix itself. As the enzymes like Taq polymerase are produced by living organisms (most often E. coli) it is quite hard to get rid of all their DNA and not destroy the enzyme. It means that with 16S targeting primers you always get some signal if you let the PCR run for long enough time (number of cycles), as Sven Reister said, over 36 cycles the chance that you get some signal is increasing. This is reason why in some 16S diagnostic tests, they recommend limiting the number of cycles to prevent false positivity.
Amplification in NTC could be due to Contamination of your reactions by DNA
Reagent contamination
Primer-dimer formation
So I would suggest you troubleshoot according to the kit manual and get clean data with negative NTC.
Hi, Termeh Hesam Mahmoudinejad
Could you please send an amp plot result for us to evaluate it with more accuracy? Have you double checked your primer design and every other step?
Waiting your answer.
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