Precellys 24 high throughput tissue homogenizer

We used a modification of Repeated Bead Beating (RBB) method that efficiently extracts bacterial community DNA based on comparative analysis of human fecal samples [23 (link)]. All frozen samples were thawed on ice and 1 ml of sterile ice-cold PBS was added, except for the left fornix flocked swabs that were stored in saline. Before the removal of the sampling device with sterile forceps the samples were vortexed for 30 secs to dislodge the bacteria and briefly spun down to avoid spills when opening the tube. An aliquot of the clarified supernatant was taken for the measurement of soluble protein content. The rest of the sample, including both the mucous pellet and the supernatant, was moved to 2 ml screw-cap tube and an equal volume of lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 4% SDS) was added. To break to cells, samples were bead beaten using a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, France) with 0.1 mm zirconium-silica beads (Biospec Products, Bartlesville, OK, USA) for 2 x 30 sec at 5500 rpm. DNA from the lysates was purified using QIAamp DNA Minikit (Qiagen, Hilden, Germany) without the lysis step according to the manufacturer’s instructions. The samples were eluted in 200 μl of kit elution buffer.