Mer omics research
Phage DNA extraction. Phage particles were isolated from 5 g aliquots of frozen stool with 50 ml Phage Buffer (10 mM Tris, pH 7.5, 10 mM MgCl2, 68 mM NaCl, 1 mM CaCl2) before homogenization by vortexing for 20 min at the highest speed (SI-H506, Horizontal 50-mL Tube Holder, Scientic Industries). Samples were centrifuged 3 times at 4 °C: 2 min at 872 x g, 10 min at 3800 x g, and 20 min at 7500 x g. After each centrifugation, supernatants were transferred to new 50 ml Falcon tubes and pellets discarded. Supernatants were ltered through 0.22 μm lters (EMD Millipore Sterivex-GP SVGPL10RC Polyethersulfone Filter Unit, Millipore). To concentrate virus particles, 10 ml of ltered supernatants were concentrated to 1 ml by centrifugation with 100 Da Amicon Ultra lters (Amicon Ultra-15 Centrifugal Filter Units, Millipore) at 3488 x g at 15 °C. Supernatants in Amicon tubes were washed two times with 5 ml Phage Buffer and volumes adjusted to 1 ml. Supernatants were ltered through 0.45 μm syringe lters (Cellulose acetate membrane syringe lter, Filter Technology) into 1.5 ml phase-lock gel tubes (5 PRIME), 40 μL lysozyme (10 mg/mL, Sigma-Aldrich) was added, and ltrates incubated for 30 min at 37 °C under shaking at 300 rpm. After incubation, 400 μL chloroform was added to samples before incubating for
15 min at room temperature with gentle inversion every 2 minutes. Samples were centrifuged at 14,000 x g for 5 min at room temperature and supernatants transferred to 1.5 ml Eppendorf tubes. A mix of 500 U bovine pancreas DNase I recombinant (Roche), 33 U Baseline-ZERO DNase (Epicentre), 6 U Salt Active Nuclease (ArcticZymes), and 500 U RNase A (Roche) was added to samples with 100 μl 10 × Incubation buffer (Roche) for incubation at 37 °C for 90 min followed by DNase inactivation at 75 °C for 10 min. After DNase/RNase treatment, phage particles were stored overnight at 4 °C. Phage DNA was extracted using Phage DNA Isolation Kits (Norgen Biotek) according to the manufacturer's protocol. DNA quantity was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientic Inc.). Phage DNA samples were stored at -80 °C.
23.10.2020 kl 19:52 384
San’s rolle i dette er at det fjerner DNA. Deretter sekvensieres bakteriene og sorteres etter type om jeg har skjønt det rett. Målet er å se endringer i bakteriefloraen etter antibiotikabruk. Det er helt rett, det er komplisert. Men om du har fulgt med så tegnet de nylig en avtale med Simcere som bruker samme prinsipp. Forskjellen er at dette er avføringsprøver mens Simcere brukte biopsier og testet på bakterier fra implantater.
Det har kommet en hel del forskning med denne metodikken og det er morsomt å se det kommersialiseres.