Optimizing Stool DNA Extraction

To evaluate the need for stool homogenisation during collection, aliquots (250 mg) of each sample were suspended in 0.1 M Tris (pH 7.5), 250 μl of 4 M guanidine thiocyanate, 40 μl of 10% N-lauroyl sarcosine and 500 μl of 5% N-lauroyl sarcosine, as previously described in [15 (link)]. DNA extraction was carried out by mechanical disruption of the microbial cell wall using beads (Lysing matrix E, MP Biomedicals, Spain). The disruption was performed by shaking the mixture using the Bead-Beater-8 (BioSpec, USA) at a medium speed of about 1500 oscillations/min for 3 minutes, followed by 3 minutes in ice and again followed by 5 minutes at a medium speed of about 1500 oscillations/min. Finally, nucleic acids were recovered from clear lysates by alcohol precipitation.
To evaluate the effect of stool water content and a bead-beating step, aliquots of samples were homogenised with various volumes of PBS (final weight of 250 mg) and with or without beads, as described in Table 1. They were then processed the same way as described above. In samples in which beads were not used, the bead-beater step was also omitted.
After genomic DNA extraction, an equivalent of 1 mg of each sample was used for DNA quantification using a NanoDrop ND-1000 Spectrophotometer (Nucliber). DNA integrity was examined by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer with the DNA 12000 kit, which resolves the distribution of double-stranded DNA fragments up to 17,000 bp in length.