Fastprep 24 5g bead beater

Extraction of genomic DNA, PCR with gene-specific primers (see Table S5 in the supplemental material), and cloning of genes into plasmids pET9a-USER-1 and pET9a-USER-2 were carried out as described previously (10 (link)). Expression of recombinant enzymes was carried out in ZYP-5052 autoinduction medium (40 (link)) supplemented with appropriate antibiotics, and cells were disrupted in a FastPrep-24 5G bead beater (MP Biomedicals) as described by Schultz-Johansen et al. (10 (link)). Recombinant, His-tagged enzymes were subsequently applied to a HisTrap FF column (GE Healthcare, Uppsala, Sweden) charged with 100 mM NiSO₄. After a washing step performed with 50 mM imidazole, the bound proteins were eluted with a linear gradient of imidazole ranging from 50 to 700 mM. Final protein concentration and purity were determined with a NanoDrop spectrophotometer (Thermo Scientific, Illkirch, France) and by SDS/PAGE, respectively. Extracts from E. coli cells treated with an empty vector were processed and analyzed in parallel as negative controls.

Approximately 500 µl of wet cell mass was mixed with 400 µl of 0.5-mm diameter acid washed glass bead (BioSpec Products Inc.) and 600 µl of breaking buffer (200 mM Tris–HCl pH 8.0, 150 mM ammonium sulfate, 1 mM EDTA and 10% v/v glycerol). The mixture was homogenized using FastPrep24™ 5G bead beater (MP Biomedicals) by beating for 30 s 8 times with a 1-min cooling interval between beatings. The lysate was first centrifuged at 7000 rpm for 20 min at 4 °C and the supernatant was collected and further clarified by 20-min centrifugation at 13,000 rpm. After concentrations were determined by Bradford assay, the protein samples were stored at − 20 °C for subsequent analysis.
For SDS-PAGE and Western blot analysis approximately 100 µg or 200 µg of total protein from each putative transformant was analyzed on a discontinuous SDS-PAGE gel consisting of 5% (v/v) stacking gel and 10% or 12% (v/v) separating gel. For samples in a denatured condition, samples were boiled for 10 min prior to the loading on the gel. For samples in a non-denatured condition, samples were loaded on the gel after being mixed with 6 × loading buffer without dithiothreitol (non-reducing 6 × loading buffer). The gels were subsequently blotted onto a 0.4 µm Nylon membrane and the target protein was detected by anti-dengue antibody (LSBio).

100 mg of fecal sample were placed in a lysing matrix E tube containing beads (MP Biomedicals Germany Gmbh, Eschwege, France) and filled with 1ml of CTAB buffer (Promega France, Charbonnières les Bains, France). Tubes were placed in a shaking heat block at 65°C, vigorously vortexed for 1 min and mixed with 40 μL of Proteinase K Solution (Promega France, Charbonnières les Bains, France). After an incubation at 70°C for 10 min, the lysates were grinded for 30 sec at 7.0 m/s six times in a Fast Prep-24 ^™ 5G bead beater (MP Biomedicals Germany Gmbh, Eschwege, France) then centrifuged at 10,000 × g for 5 min. A volume of 300 μL of clear lysate was transferred to a tube containing 300 μL of Lysis buffer. RNA extraction was performed with the Maxwell RSC PureFood GMO and Authentication kit using a Maxwell RSC instrument (Promega France, Charbonnières les Bains, France), according to the manufacturer’s instructions. RNA was eluted in a final volume of 100μL and stored at < −70°C.
Negative (non-template control) and positive (hedgehog betacoronavirus) controls (RNA extraction control) were performed for each set of 16 samples tested.

S. lophii spores were washed and resuspended in sterile BS100 buffer (25 mM HEPES, 100 mM potassium acetate, 15 mM magnesium acetate and 1 mM DTT). Spores were disrupted using a Fastprep-24 5 G bead beater (MP Biomedicals, Fisher Scientific) and 0.1 mm glass beads. Cell debris and non-disrupted spores were pelleted by centrifugation at 9,000g for 15 min at 4 °C. The supernatant was collected and layered on top of a 30% sucrose cushion in BS100 buffer and centrifuged at 206,000g for 3 h at 4 °C. The supernatant was discarded, and the pellet was resuspended by gentle shaking for 1 h at 4 °C in BS100 buffer. The crude extract was layered over a continuous sucrose gradient (10–40%) in BS100 buffer and centrifuged at 125,000g for 80 min at 4 °C. Fractions were collected and analysed for RNA using a Nanodrop (Thermo Scientific) at 260 nm. Vivaspin columns (Sartorius) with 100 kDa cut-off were used to remove the excess of sucrose and concentrate the sample to a concentration between 5 and 15 mg ml⁻¹.