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Old 04-22-2019, 12:57 PM   #1
Junior Member
Location: Canada

Join Date: Apr 2019
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Default no PCR band

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?
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Old 04-22-2019, 02:12 PM   #2
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PCR inhibitors?
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Old 04-23-2019, 04:42 AM   #3
Location: Canada

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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.

Sasaki, Y., Miyoshi, D., & Sugimoto, N. 2006. Effect of molecular crowding on DNA
polymerase activity. Biotechnology Journal 1: 440–446.

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
serum albumin. Biochemical Journal 112: 619.
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16s library, amplicon pcr, illumina, illumina 16s protocol, metagenomics

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