Fastprep 24 classic

Stored plasma samples were melted and diluted/extracted with three-fold of ACN/water solution (84/16, v/v) and shaken on an orbital shaker for 15 min at RT. Extracts were centrifuged (RT, 5 min, 8.000× g), and supernatants were collected and subsequently diluted with 0.01M PBS, pH = 7.4. The dilution factor on supernatant was 250×.
Usually, 0.25 g of excised organs/samples was homogenized in ice-cold, 0.5 mL of 50 mM sodium acetate buffer (pH = 4.80). First, homogenization was performed with a bench-top bead beating lysis system (FastPrep-24 Classic, MP Biomedicals, Irvine, CA, USA) using metal beads followed by incubation for 3 h at 37 °C on a shaker with the addition of β-glucuronidase/arylsulfatase from Helix pomatia, accordingly to the instructions of the manufacturer. Following the incubation, the homogenized sample was diluted with ACN/water solution (84/16, v/v) and shaken (extracted) for 15 min. Next, extracts were centrifuged (10.000× g, 10 min, at 4 °C), and the supernatant was separated and subsequently diluted 100× with 0.01 M phosphate-buffered saline (PBS, pH = 7.40). Finally, the diluted extracts were used for measurements with the assay.

Cell lysis was achieved using a customized cordless oscillating multifunctional power tool (Einhell Varrito, EAN: 4006825618648). Fitted to the power tool was an adapted blade that held a 15 mL nylon centrifuge tube, into which lysis tubes were inserted and packed, so there was no movement independent of the outer tube (see Figure 8A,B). The device, referred onward as the “Osci-lyser”, delivered a rotational movement of 3.2°, and its adapted blade provided an 8.5 cm throw, resulting in approximately 5 mm movement on each oscillation. It had a variable speed selector, ranging from 183 to 333 Hz. The lowest speed was used for 20 s to achieve cell lysis in the study. After lysis, the sample was centrifuged for 1 min at 8000 rpm using the Bento lab before a 13.2 µL aliquot was taken for RPA amplification.
To test the efficacy of the Osci-lyser, a comparison was made against a bench top laboratory device, specifically, the MP biomedicals Fastprep-24 classic. This was achieved by performing triplicate DNA extractions on cultures of Alexandrium, approximately 50,000 cells in 400 µL of distilled water for each extraction. The fast prep was set to 4.0 M/S and run for 20 s. The Osci-lyser had the speed set to minimum, and a sample was lysed for 20 s. Once lysis was complete, samples were centrifuged, and 10 µL from each sample was quantified using a Qubit fluorometer.

E. coli was grown in LB culture medium at 37 °C with constant agitation (180 rpm). After reaching an exponential phase (OD₆₀₀ ~ 0.6), cells were washed twice with Borax-Citrate buffer 15 mM (pH 9.4). After that, the cells were homogenized three times using glass beads and the FastPrep-24™ Classic (MP Biomedicals, LLC, Irvine, USA) for 1 min at 4 m/s. Subsequently, the samples were centrifuged for 3 min at 27,670 x g and the synthesis of nanoparticles was evaluated in the conditions described above. Protein concentration of the lysate was determined by a Quick Start™ Bradford Protein Assay (Bio-Rad Inc., Hercules, CA, USA).

In 2018, 35 ticks were collected from 29 patients with a history of tick bites in Gwangju Metropolitan City, Jeollanam Province, ROK. Ticks were identified on the basis of their molecular, morphological, and standard taxonomic characteristics. Briefly, the ticks were first decontaminated using 70% ethanol, rinsed twice using sterile phosphate-buffered saline (PBS), and dried on sterile filter paper. Each sample was then placed in a hard-tissue-grinding MK28 tube (Bertin Technology, Rockville, MD, USA) containing 800 µl PBS and 1× PC/SM (i.e., penicillin and streptomycin). Subsequently, ticks were ground using a FastPrep-24 Classic instrument (MP Biomedicals, Solon, OH, USA) and were stored at − 80 °C until DNA extraction.

Mtb cell lysis was performed by four rounds of bead beating (40 s) in FastPrep-24 Classic bead beating grinder (MP Biomedicals, Irvine CA, USA) using glass beads in a total volume of 1 ml of lysis buffer with intermittent chilling on ice. The lysis buffer comprised 50 mM tris-HCl, 150 mM NaCl, 5% glycerol, 0.5 mM β-mercaptoethanol, and 1× cOmplete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). For Western blotting, 50 μg of total cell lysate was resolved on a 12% SDS-PAGE gel and transferred to immunoblot polyvinylidene difluoride membrane in a Mini Trans-blot Cell (Bio-Rad, Hercules, CA, USA) at a constant current of 350 mA. Cbs was detected using Cbs antisera (anti-rabbit; 1:10,000) and anti-rabbit immunoglobulin G horseradish-linked antibody (1:10,000; Cell Signaling Technology, Danvers, MA, USA). Rho (gift from V. Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science) was used as internal control (anti-rabbit; 1:10,000). The blots were developed using Clarity Max ECL Western blotting substrate in a GelDoc XR+ Gel Documentation system. Densitometry was performed by plotting band intensities quantified using ImageJ (https://imagej.nih.gov/ij/download.html).

Samples of sediment and biofilm from all treatments and time points were thawed at room temperature and used for both RNA and DNA extraction using PowerSoil^® Total RNA Isolation Kit and RNA Powersoil^® DNA Elution Accessory Kit, respectively (MoBio Laboratories, Solana Beach, CA, USA) following manufacturer’s instructions and including an homogenization step (3x) by bead-beating (FastPrep^®-24 Classic, MP Biomedicals, CA, USA). Water samples from blank controls were also thawed at room temperature and filtered through 0.22 μm pore size 47-mm diameter polycarbonate filters (ISOPORE, Millipore, MA, USA) that were further used for DNA and RNA extraction as described for sediment and biofilm samples. Concentration of DNA and RNA in resulting extracts was determined using QUBIT^® 2.0 Fluorometer (Invitrogen Molecular proves Inc., Oslo, Norway). TURBO DNAfree^™ Kit (Ambion, Austin, TX, USA) was used to remove DNA traces in RNA extracts. Reverse transcription of RNA to cDNA was done using random hexamer primers and Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Concentration of cDNA was also quantified using Qubit^® 2.0 Fluorometer.

Total genomic DNA was isolated from cacao leaves using the Plant DNeasy kit (Qiagen) according to manufacturer’s recommendations. Eighty milligrams of fresh leaves were ground with liquid nitrogen in a microcentrifuge tube in the presence of ceramic beads using a FastPrep-24 Classic (MP Biomedicals) homogenizer. Alternatively, five hundred milligrams of cacao leaves were frozen in liquid nitrogen and ground to a fine powder, which was then mixed with 5 mL of extraction buffer [100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 8, 1.4 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 2% w/v mixed alkyltrimethylammonium bromide, 1% w/v PEG6000 and 0.5% w/v Na₂SO₃ added freshly]. Samples were incubated at 74 °C for 30 min with 2 mg/mL RNase (Qiagen), extracted twice by 5 mL of chloroform–isoamyl alcohol (24:1) and precipitated with 5 mL of isopropanol at − 20 °C. DNA pellet was rinsed with EtOH and resuspended in 500 μL of sterile distilled deionized water. After quantification, DNA quality was assessed by PCR using the microsatellite mTC 351 primer pair (Table 2)^{37 (link)}.