B. subtilis and B. anthracis cells were grown overnight at 37 °C and inoculated to an OD600 of 0.05 in 50 ml LB. The LB media was supplemented with 1 mM IPTG for the EF-P complementation strain. Log phase culture (~OD 0.6) was collected by centrifugation at 8000 rpm for 10 minutes (Beckman Coulter Avanti J-15R, rotor JA-10.100). The pellets were resuspended in 200 μl gradient buffer containing 20 mM Tris (pH 7.4 at 4°C), 0.5 mM EDTA, 60 mM NH4Cl, and 7.5 mM Mg2 (55 (link)). The cell resuspension was lysed using a homogenizer (Beadbug6, Benchmark) by four 30 s pulses at speed 4350 rpm with chilling on ice for 3 min between the cycles and clarified by ultracentrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24×2). Clarified cell lysates were normalized to 1500 ng/μl and loaded onto 10 – 40% sucrose gradients in gradient buffer. Ultracentrifugation separated ribosome species at 30,000 rpm for 3 hours at 4μC (Beckman Coulter, rotor SW-41Ti). Gradients were collected using a Biocomp Gradient Station (BioComp Instruments) with A260 continuous readout (Triax full spectrum flow cell). The area under each peak for 30S, 50S, and 70S and the several peaks for polysomes, was quantified using Prism (GraphPad).
Beadbug 6
Manufactured by Benchmark Scientific
Sourced in United States
The BeadBug 6 is a homogenizer designed for efficient disruption and lysis of biological samples. It utilizes high-speed agitation of sample-containing tubes with metal or ceramic beads to physically disrupt tissues, cells, and microorganisms. The BeadBug 6 is capable of processing multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Avanti j 15r, by Beckman Coulter (1 mentions)
Biocomp gradient station, by BioComp Instruments (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
Zirconium beads, by Benchmark Scientific (1 mentions)
Janus automatic pipetting workstation, by PerkinElmer (1 mentions)
Enspire plate reader, by PerkinElmer (1 mentions)
Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions)
Nuclei lysis solution, by Promega (1 mentions)
Prism 8 v8, by GraphPad (1 mentions)
Iscript advanced cdna kit, by Bio-Rad (1 mentions)
Nano glo substrate, by Promega (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Tissue extraction reagent 1, by Thermo Fisher Scientific (1 mentions)
Enzyme linked immunosorbent assay kit, by R&D Systems (1 mentions)
5 protocols using beadbug 6
1
Ribosome profiling of B. subtilis and B. anthracis
2
Homogenization and Analysis of Whole Milk
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Whole milk (500 µL) samples were thawed and transferred into a homogeniser tube containing zirconium beads (3.0 mm diameter, D1032-30, Benchmark Scientific, Sayreville, NJ, USA). The milk samples were homogenised for 3 cycles at 2500 rpm, 5 s per cycle with a 3 s pause using a microtube homogeniser (BeadBug 6, Benchmark Scientific, Sayreville, NJ, USA). The assays for macronutrient and metabolic hormone measurements (all previously validated for use in human milk) were performed using a JANUS automatic pipetting workstation (PerkinElmer, Inc., Waltham, MA, USA) and measured on a EnSpire plate reader (PerkinElmer, Inc., Waltham, MA, USA).
3
Plasmid Integration Locus Determination
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Cells from 1 mL bacterial cultures were collected by centrifugation, washed three times with 7H9 + Tween80 (to remove free phage from lysogenic cultures), resuspended in 0.6 mL nuclei lysis solution (Promega), and transferred to tubes containing Lysis matrix B beads (MP Biologicals). Cells were milled at 4,300 rpm for 1 min in a Benchmark Scientific BeadBug6 three times, cooling on ice for 2 min between times. The cell lysate was treated with RNAseA before adding 0.6 mL phenol-chloroform-isoamyl alcohol 24:23:1 (Thermo). Tubes were inverted to mix and centrifuged to separate aqueous and organic phases; the aqueous phases were collected and re-extracted similarly. DNA was precipitated from the final aqueous phase with isopropyl alcohol and 3 M sodium acetate; the precipitated DNA was collected by centrifugation, washed with 75% ethanol, air dried, and resuspended in 50–100 μL of 5 mM Tris-HCl pH 8.0. Illlumina sequencing was used to determine the integration locus of plasmid pCG46 in M. smegmatis.
4
SARS-CoV-2 Viral Quantification in Murine Tissues
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Indicated organs (nasal cavity, brain and lungs) from infected or uninfected mice were collected, weighed, and homogenized in 1 mL of serum-free RPMI media containing penicillin-streptomycin and 1.5 mm Zirconium beads with a BeadBug 6 homogenizer (Benchmark Scientific, T Equipment Inc). Viral titers were measured using two highly correlative methods. First, the total RNA was extracted from homogenized tissues using RNeasy plus Mini kit (Qiagen), reverse transcribed with iScript advanced cDNA kit (Bio-Rad) followed by a SYBR Green Real-time PCR assay for determining copies of SARS-CoV-2 N gene RNA using primers are listed in Table S3 .
Second, we used nanoluciferase activity as an efficient surrogate for a plaque assay. Dilutions from infected cell homogenates were applied on Vero E6 monolayer. 24 hour post infection, infected Vero E6 cells were washed with PBS, lysed with Passive lysis buffer and transferred into a 96-well solid white plate (Costar Inc) and nanoluciferase activity was measured using Tristar multiwell Luminometer (Berthold Technology) for 2.5 seconds by adding 20 μl of Nano-Glo® substrate in nanoluc assay buffer (Promega Inc). An uninfected monolayer of Vero E6 cells treated identically served as controls for determining background and obtain normalized relative light units. The data were processed and plotted using GraphPad Prism 8 v8.4.3.
Second, we used nanoluciferase activity as an efficient surrogate for a plaque assay. Dilutions from infected cell homogenates were applied on Vero E6 monolayer. 24 hour post infection, infected Vero E6 cells were washed with PBS, lysed with Passive lysis buffer and transferred into a 96-well solid white plate (Costar Inc) and nanoluciferase activity was measured using Tristar multiwell Luminometer (Berthold Technology) for 2.5 seconds by adding 20 μl of Nano-Glo® substrate in nanoluc assay buffer (Promega Inc). An uninfected monolayer of Vero E6 cells treated identically served as controls for determining background and obtain normalized relative light units. The data were processed and plotted using GraphPad Prism 8 v8.4.3.
5
Colon Tissue Cytokine Analysis
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The colon tissue in Tissue Extraction Reagent I (Invitrogen, Waltham, MS, USA) was homogenized using BeadBug™ 6 (Benchmark Scientific, Sayreville, NJ, USA). The homogenates were centrifuged (10,000× g for 10 min at 4 °C), and a supernatant was used. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-6 in colon tissue were analyzed by enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN, USA). The absorbance was measured at 450 nm (BioTek).
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