I extracted DNA from some kombucha products with a manual protocol, with one final step using phenol, for 16S and ITS sequencing.
However, there is a problem for the majority of my samples in PCR step. Apparently, there is/are some inhibitors in my samples that impede PCR. My samples pH is around 3 and they contain polyphenols.
Did anyone have any similar experience or any suggestion?
Hi Atena,
I am not sure how concentrated your samples are, but for inhibitors of PCR often dilution is the solution. If you can dilute and still have adequate input DNA to feel confident you will have a good representation of your community put into the amplification then I suggest trying that first, its the easiest.
Frank
CTAB dna extraction has often been used when high levels of polyphenols are present. I agree entirely with Frank that diluting the dna ( and therefore the inhibitors) and possibly adding a couple of extra cycles of pcr is the simplest solution
The Benzyl chloride method for DNA extraction aslo can solve your problem.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310651/
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