For DNA extraction, GBS colony material was collected with a calibrated loop (1 µl) and suspended in 600 µl nuclease free water (Sigma-Aldrich, St Louis, MO, USA). The suspension was mixed with 0.1 mm silica beads (BioSpec Products Inc., Bartlesville, USA) and added to the FastPrep24 (MP Biomedicals LLC, Irvine, CA, USA) and then run at 6.5 m/s for three 2-minute cycles. DNA was extracted from 200 µl samples using the IndiMag Pathogen kit (Indical Bioscience GmbH, Leipzig, Germany) and eluted in nuclease free water. DNA concentration was measured using the Invitrogen Qubit 3.0 Fluorometer (ThermoFisher Scientific Inc., Waltham, MA, USA), and adjusted to 7.5 ng/µL. Library preparation and whole genome sequencing were performed by Clinical Genomics, Science for Life Laboratory (Clinical Genomics, Solna, Sweden) on the Illumina NovaSeq (Illumina, Inc. CA, US) resulting in paired end reads of 150 bp in length.
0.1 mm silica beads
Manufactured by Biospec
Sourced in United States
The 0.1 mm silica beads are a type of laboratory equipment used for various applications. They are made of silica and have a diameter of 0.1 millimeters. The core function of these beads is to serve as a material for various laboratory procedures and experiments, where their small size and specific properties may be required.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (9 mentions)
Nuclease free water, by Merck Group (3 mentions)
Fastprep 24, by MP Biomedicals (3 mentions)
Direct zol rna miniprep plus, by Zymo Research (3 mentions)
Qubit dsdna br protein assay kit, by Thermo Fisher Scientific (2 mentions)
Novaseq, by Illumina (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
3xflag peptide, by Merck Group (1 mentions)
Fastprep 24 5g instrument, by MP Biomedicals (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Mini beadbeater 96, by Biospec (1 mentions)
Trace mineral supplement, by American Type Culture Collection (1 mentions)
Trace vitamin supplement, by American Type Culture Collection (1 mentions)
D maltose, by Merck Group (1 mentions)
M9 salts, by Teknova (1 mentions)
Sterile filtered fetal bovine serum, by Merck Group (1 mentions)
Vitamin k1 hemin solution, by BD (1 mentions)
D fructose, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
10 protocols using 0.1 mm silica beads
1
DNA Extraction and Whole Genome Sequencing
2
Immunoprecipitation of FLAG-tagged Protein
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WalR-FLAG was immunoprecipitated from 25 ml (Phos-tag experiment) or 600 ml (mass spectrometry experiment) cultures at OD600~0.4 using anti-FLAG M2 magnetic beads (Sigma M8823) according to the manufacturer’s instructions with the following modifications. Initial lysis to break down the cell wall was performed in 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 μg/ml DNaseI, 3 mM MgCl2, 1x Halt phosphatase inhibitor cocktail (Pierce), 1 mM PMSF, and 1 mg/ml lysozyme. After 20 min of lysis on ice with periodic vortexing, cell pellets were spun down and resuspended in lysis buffer without lysozyme and applied to 0.1 mm silica beads (BioSpec, 11079101z) in pre-chilled screw cap tubes. Cells were lysed using a FastPrep-24 5G instrument (MP Biomedicals) using 4 runs of 6.5 m/s for 40 seconds with samples placed on ice for 3 min between each run. Lysates were cleared by centrifugation at 19,000 x g for 20 min at 4°C. Elutions from the anti-FLAG M2 magnetic beads were performed competitively by addition of the 3x-FLAG peptide (Sigma F4799).
3
DNA Extraction from Epithelial Tissue
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Tissue samples were processed for total DNA using a protocol adapted from Roussel et al. (2005) (link) and purified using the hot phenol method described in Dill-McFarland et al. (2017) (link). In short, 0.25 to 0.5 g of epithelial tissue was subsampled from biopsies using sterile tissue shears and combined with 1 mL of chilled DNA extraction buffer [100 mM Tris-HCl, 10 mM EDTA, 0.15 M NaCl (pH 8.0)] and a 4-mm silica bead (BioSpec Products Inc., Bartlesville, OK). Samples were vortexed on high 5 times for 30 s each using a GeneMate Vortex Mixer (BioExpress, Kaysville, UT). The resulting supernatants were combined with phenol and 20% SDS and mechanically disrupted using 0.1-mm silica beads (BioSpec Products Inc.) and a Mini-Beadbeater 96 (BioSpec Products Inc.). Samples were then incubated at 60°C and subjected to another round of bead beating. Aqueous-phase crude extracts were then washed twice with phenol: chloroform: isoamyl alcohol (25:24:1) and precipitated overnight at -20°C with 2 M sodium acetate and isopropanol. Cell pellets were washed with cold ethanol, dried, and suspended in 20 μL of Tris-EDTA buffer. DNA was quantified using a Qubit Fluorometer (Invitrogen, San Diego, CA) and stored at -80°C until library preparation.
4
Transcriptomic Analysis of B. thetaiotaomicron
5
Whole Genome Sequencing of GBS Isolates
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Extraction of DNA was carried out from all confirmed GBS isolates (n = 58), using a magnetic bead-based method. A calibrated loop (1 μL) was used to suspend colony material in 600 μL nuclease free water (Sigma-Aldrich, St Louis, MO, USA) and mixed with 0.1 mm silica beads (BioSpec Products Inc., Bartlesville, USA). The suspension was added to the FastPrep24 (MP Biomedicals LLC, Irvine, CA, USA) and run at 6.5 m/s for three 2-minute cycles. DNA was extracted from 200 μL samples using the IndiMag Pathogen kit (Indical Bioscience GmbH, Leipzig, Germany) and eluted in nuclease free water. The Invitrogen Qubit 3.0 Fluorometer and the Qubit dsDNA BR Protein Assay kit (ThermoFisher Scientific Inc., Waltham, MA, USA) were used to measure DNA concentrations, which were adjusted to 7.5 ng/μL. Library preparation and whole genome sequencing were performed by Clinical Genomics, Science for Life Laboratory (Clinical Genomics, Solna, Sweden) on the Illumina NovaSeq (Illumina, Inc. CA, US), resulting in paired-end libraries of 150 bp read length.
6
Extraction of Protein from S. mutans
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Overnight THYE cultures of S. mutans UA159 and its derivates were diluted 1:25 in 100 ml BTR-G medium ± environmental stress and incubated anaerobically until reaching mid-log growth phase. The cells were harvested by centrifugation (Eppendorf 5810R, 10 min, 3200 × g, 4 °C), washed twice in phosphate-buffered saline (PBS) buffer (pH 7.4), and then resuspended in 500 µl PBS buffer (pH 7.4) containing 0.2 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine. The suspension was transferred to a 2 ml screw-cap tube containing 1 ml of 0.1 mm silica beads (Biospec) and then lysed in an Omni Bead Ruptor 24 via six 15 s homogenization cycles set at 5 m/s with 1 min incubation periods on ice between cycles. The homogenate was centrifugated for 20 min at 16,000 × g in a table top centrifuge at 4 °C and the supernatant was used as the cell lysate for downstream applications. Protein concentrations were determined by Bradford assay using the Protein Assay Dye Reagent (BioRad). Standard curves were created using a bovine serum albumin (Sigma) standard.
7
Whole Genome Sequencing of GBS Isolates
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Extraction of DNA was carried out from all confirmed GBS isolates (n=58), using a magnetic beadbased method. A calibrated loop (1 µL) was used to suspend colony material in 600 µL nuclease free water (Sigma-Aldrich, St Louis, MO, USA) and mixed with 0.1 mm silica beads (BioSpec Products Inc., Bartlesville, USA). The suspension was added to the FastPrep24 (MP Biomedicals LLC, Irvine, CA, USA) and run at 6.5 m/s for three 2-minute cycles. DNA was extracted from 200 µL samples using the IndiMag Pathogen kit (Indical Bioscience GmbH, Leipzig, Germany) and eluted in nuclease free water. The Invitrogen Qubit 3.0 Fluorometer and the Qubit dsDNA BR Protein Assay kit (ThermoFisher Scientific Inc., Waltham, MA, USA) were used to measure DNA concentrations, which were adjusted to 7.5 ng/µL. Library preparation and whole genome sequencing were performed by Clinical Genomics, Science for Life Laboratory (Clinical Genomics, Solna, Sweden) on the Illumina NovaSeq (Illumina, Inc. CA, US), resulting in paired-end libraries of 150bp read length.
8
Transcriptomic Analysis of B. fragilis
9
Transcriptomic Analysis of B. fragilis
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Triplicate cultures of B. fragilis ATCC 25285 were diluted 1:100 from overnight cultures into 23 mL of BHI medium supplemented with 1% Vitamin K1-hemin and complex BHI medium and grown to early exponential phase (OD600 0.1–0.3) in anaerobic conditions. Cultures were divided into 6 mL aliquots in 50-mL Falcon tubes and DMSO (0.04%) or LEA (25 μM) or AEA (25 μM) was added to one aliquot from each triplicate culture. After 10 minutes, 1 mL of each condition was pelleted in the anaerobic chamber, the supernatant discarded and the pellet resuspended in 500 μL Trizol. Trizol suspensions underwent a step of bead-beating using ~500 μL of 0.1 mm silica beads (BioSpec). RNA was extracted with the Direct-Zol RNA MiniPrep Plus (Zymo Research) according to manufacturer’s instructions.
10
Homogenization and Protein Extraction
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Whole organs from one animal and cells from pooled animals were first washed in PBS followed by a homogenization step using a Polytron PT 2100 Homogenizer (Kinematica, Luzern, Switzerland). From each homogenized organ and cell, 250 μl samples were mixed with 100 mg 0.1 mm silica beads (Biospec Products, Bartlesville, OK, USA) and further homogenized using a FastPrep-96 instrument (1,600g, 180 s) (MP BIOMEDICALS, Santa Ana, CA, USA). The silicon beads were removed with a centrifugation step (14,000g, 1 min) and the protein concentration in the supernatant was determined with Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
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