DNeasy Blood & Tissue Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Experimental Protocol
DNA from all samples was extracted using a Qiagen DNeasy Extraction kit according to the manufacturer's instructions and the concentration was assessed using Nanodrop. qPCR (Applied Biosystems ViiA7) was performed on these samples using a previously published 16S rRNA assay [1] that utilized a standard curve of a serial dilution of Escherichia coli (cycling conditions were 1 cycle at 95°C for 15 minutes, 40 cycles at 95°C for 15 seconds and 60°C for 1 minute, followed by a standard denaturation curve protocol). The assay was a SYBR green qPCR assay and three replicates were used per sample. Data were collected using the ABI ViiA7 RUO software program. The same assay was adapted to ddPCR (Bio-Rad QX200) and the experiments were performed using the following protocol: 1 cycle at 95°C for 5 minutes, 40 cycles at 95°C for 15 seconds and 60°C for 1 minute, 1 cycle at 4°C for 5 minutes, and 1 cycle at 90°C for 5 minutes all at a ramp rate of 2°C/second. Bio-Rad's T100 thermal cycler was used for the PCR step. No standard curve was required for the ddPCR and the droplets were quantified using the Bio-Rad Quantisoft software. A total of two replicates were used per sample. A threshold cutoff of 20000 was chosen based on preliminary experiments, which accurately separated positive from negative droplets. For both protocols, negative controls that comprised of DNase and RNase free water were used and were run alongside the samples.

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Manufacturer protocol

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