Currently, I am working with primers which amplify 16S rRNA gene, included in all bacteria genome and highly conservative. Given that commercial polymerases are synthetized in bacteria (E. coli mainly), part of the gDNA remain even after purification, none guaranteed 100% gDNA free polymerases.
Primers are designed to amplify the 16s gene of all bacteria, thus amplify residual gDNA from commercial kit in NTC. Is there any possibility of extracting this signal from the final outcome of the qPCR (either SYBR Green or FAM-probe)?
You could run a negative control PCR adding water instead of your sample to determine how strong is the signal given by the residual 16S rRNA. In case you get any amplification, you could just ignore the same signal in the samples to test.
I have done it already, non-template control, negative controls just with water and with the commercial enzyme by itself. The main problem is that I need to remove the signal because I am using the 16s as housekeeping gene, this residual contamination does not appear in the study gene because requires very specific primers. If I do not remove the signal, I will underestimate the signal of the gene of study.
If you want to romove all gDNA you could perform a DNase I treatment then. I think the attached paper could help you with it, they treat a PCR Master Mix to remove DNA.
Hope this helps you somehow!
http://jcm.asm.org/content/41/4/1763.full
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