no PCR band

[Paradise]

Hi,
I have some DNA samples of water and ice and I want to prepare 16s library.
3 samples out of 13 don't have any bands after amplicon PCR. Their DNA concentrations are 19.1, 14.7, and 15.2 ng/ml measured by Qubit. I was able to see bands for other samples with lower DNA concentrations.
Does anybody have any idea?
Thanks

[luc]

PCR inhibitors?

[ATϟGC]

A few questions that might help: What DNA polymerase are you currently using for PCR? Are your 16S primers tailed for high-throughput sequencing?

If inhibition is the problem, dilution will often solve it. I would try a 1 in 10 as well as a 1 in 100 dilution of your DNA extracts. In rare cases I have had to resort to a 1 in 1000 dilution to achieve amplification.

If that does not work I would then try including one or more of the following PCR enhancers: BSA, PEG 8000 and Trehalose. Some PCR mastermixes already include these additives. Another option is using a DNA polymerase that is more robust to inhibition.


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Speiss, A., T. Mueller, & Ivell, R. 2004. Trehalose is a potent PCR enhancer: lowering of
DNA melting temperature and thermal stabilization of Taq polymerase by the
disaccharide trehalose. Clinical chemistry 50: 1256-1259.

Pierpoint, W. S. 1969. o-Quinones formed in plant extracts. Their reaction with bovine
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