The library construction included the DNA extraction protocol that follows the recommended procedure of the DNeasy Plant Mini kit (Qiagen, Hilden, Germany), including three bead-beating steps for 3 min in a FastPrep-24 bead beater (MP Bio, Solon, OH, United States) (Lleixà et al., 2018 (link)). Extracted DNA quantity was checked by nanodrop and sent to CRG (Centre for Genomic Regulation, Barcelona, Spain). The DNA quality was checked by the Agilent 2100 Bioanalyzer, and the quantity was adjusted to 10 μg per sample in order to be sequenced by Illumina MiSeq 2x300, using the primers 341F/785R for the 16S amplicon (Herlemann et al., 2011 (link)) and ITS2F/ITS2R for the ITS amplicon (White et al., 1990 (link)).
Fastprep 24 bead beater
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep-24 bead beater is a laboratory instrument designed for the efficient disruption and homogenization of biological samples. It utilizes rapid agitation of samples along with specialized beads to physically break down and extract the contents of cells, tissues, or other materials.
Automatically generated - may contain errors
Lab products found in correlation
Lysing matrix b, by MP Biomedicals (4 mentions)
Quant it dsdna assay kit, by Thermo Fisher Scientific (3 mentions)
Qiacube, by Qiagen (3 mentions)
Ribozol, by Avantor (2 mentions)
Dneasy powersoil htp 96 dna isolation kit, by Qiagen (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Promega (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Direct zol rna isolation kit, by Zymo Research (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Promega (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Plant mini kit, by Qiagen (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (2 mentions)
Superwest pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Lysing matrix c beads, by MP Biomedicals (2 mentions)
Lysing matrix e, by MP Biomedicals (2 mentions)
Dneasy 96 powersoil pro qiacube ht kit, by Qiagen (2 mentions)
52 protocols using fastprep 24 bead beater
1
DNA Extraction and Sequencing Protocol
2
Immunoblot Analysis of Fission Yeast Asp1 Protein
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S. pombe asp1Δ cells bearing pTIN plasmids were grown at 30°C in Leu− ePMG liquid medium with 15 μM thiamine until A600 reached 0.6 to 0.9. Aliquots (8 A600 units) of cells were collected by centrifugation and resuspended in 200 μL 20% trichloroacetic acid, then supplemented with 0.7 g of 0.5 mm Zirconia beads (Biospec) and subjected to six 30-s cycles of treatment with a FastPrep-24 bead-beater (MP biomedical) at 6.5 m/s. Total acid-insoluble protein was recovered by centrifugation. The pellets were washed twice with ethanol, then air dried and resuspended in 300 μL 0.5 M Tris-HCl (pH 8.0). The samples were adjusted to 2% SDS, 10% glycerol, 10% β-mercaptoethanol and heated at 95°C for 5 min. Cell debris and insoluble material were removed by centrifugation. Aliquots of supernatant proteins (representative of 0.36 A600 units of cells) were resolved by electrophoresis through 8% polyacrylamide gels containing 0.1% SDS. Gel contents were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). The blots were probed with affinity-purified rabbit polyclonal anti-Asp1 protein. Parallel blots were probed with anti-Spt5 antibody as a loading control. Immune complexes were detected using horseradish peroxidase-linked anti-rabbit IgG (Cytiva NA934V) and an ECL (enhanced chemiluminescence) Western system (Cytiva) and visualized with an ImageQuant 800 apparatus (Amersham).
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3
Isolation and Cultivation of Sulfur-Oxidizing Microbes
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The original strain was isolated from the sulfidic water column of Solar Lake, Israel (Cohen et al., 1975b (link)), and was kindly provided by A. Oren for culturing. A monoalgal culture was grown in modified Chu’s 11 in Turks Island Salts medium at room temperature (average 22.0°C) and ambient light in a 125 mL Erhlenmeyer flask. We extracted whole community DNA using the MPBio FastDNA SpinKit and Fastprep-24 Bead Beater (MP Biomedicals, Solon, OH, USA) following the default protocol, except that 0.3 g of beads were used for bead beating. DNA was quantified using Quant-IT PicoGreen (Invitrogen, Grand Island, NY, USA) and submitted to the University of Michigan DNA Sequencing Core for library preparation and Illumina HiSeq 2 × 100 bp paired-end sequencing.
4
Analysis of Whole-Cell RHA1 Lipid Content
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For whole RHA1 lipid content analysis, cells were harvested during the transition from exponential to stationary growth phases, washed with a 150 mM NaCl and suspended in 50 mM Tris-HCl, pH 7.0, and 300 mM NaCl buffer. Cells were subjected to four rounds of 60 s of bead beating using a FastPrep-24 bead beater (MP Biomedicals, Solon, OH) set to 5.5. The sample was incubated for 5 min on ice between rounds. To identify lipids, samples of whole cells or isolated LDs were extracted with chloroform-methanol (2:1, v/v). Aliquots were analyzed by thin layer chromatography (TLC) on 60F254 silica gel plates (Merck) using hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as a solvent system. Triolein (Sigma-Aldrich, T7140) was used as the TAG standard. The plates were dried at room temperature for 10 min and immediately sprayed with a 10% cupric sulfate in 8% phosphoric acid solution. The plates were then incubated in an oven at 150 °C for 10 min.
5
Quantitative Analysis of Hippocampal Gene Expression
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Frozen hippocampal tissues were thawed and homogenised with RiboZol (VWR, UK) for 3 × 20 s in a Fast Prep-24 Bead Beater (MPBIO, Santa Ana, CA, USA) with cooling on ice between each round. RNA was extracted and quantitative PCR (qPCR) was performed as described elsewhere [22 (link)] using the gene-specific oligonucleotide primers shown in Supplementary Table S3 . mRNA expression levels in relation to the untreated controls were determined using 2−ΔCt, where ΔCt represents the difference between the threshold cycle (CT) for each target gene and the housekeeping gene (β-actin).
6
Stool DNA Extraction Protocol
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Stool was weighed, and DNA extracted using the DNeasy PowerSoil HTP 96 DNA Isolation kit (Qiagen, Chadstone VIC, Australia; Cat No. 12888–100). The following modification to the manufacturer’s instructions were employed: samples and solution C1 were added into bead tubes and heated for 10 min at 65 °C, prior to two cycles of bead beating at 6.5 m/s for 1 min using a FastPrep-24 bead beater (MP Biomedicals, Santa Ana, CA, USA). Quant-IT dsDNA Assay kit (Life Technologies, Carlsbad, CA, USA) was used to quantify DNA concentration after extraction. Extracted DNA was stored at − 20 °C prior to further analysis.
7
Efficient DNA Extraction for Primer Testing
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For testing primer efficiency, DNA was extracted from J2 CN individuals following the lysis method [37 (link)]. For screening experiments and standard curves, solutions containing a known number of posterior ends of CN females were placed in bead-beating tubes and extracted using a DNeasy Plant Pro Kit (Qiagen, Hilden, Germany), as specified by the most recent available manufacturer instructions (August 2019), with the following modifications: 50 µL of solution PS was added together with solution CD1, and homogenization was performed at 6.0 m/s for 180 s on a Fast-Prep-24 bead beater (MP Biomedicals, CA, USA), followed by 90 min digestion at 55 °C with 16 U of proteinase K (New England Biolabs Inc., Beverly, MA, USA). A subsequential 60 s bead-beating step was performed before the first centrifugation step (12,000× g for 2 min). DNA was eluted in 100 µL of nuclease-free water. Samples from the host-screening experiment were diluted 1:10. DNA quality and concentration were assessed in an ND-1000 full spectrum UV–Vis spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
8
DNA Extraction from Environmental Samples
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DNA was extracted from half of each filter using a modified Miller protocol32 (link). Half of each filter was placed into a Lysing Matrix E tube (MP Biomedicals) along with 300 µl of Miller Phosphate buffer, 300 µl of Miller SDS lysis buffer, and 600 µl of Phenol:Chloroform:Isoamly alcohol (25:24:1). A process blank was also setup and subjected to all following steps of extraction without a filter. The tubes were homogenized in a FastPrep-24 bead-beater (MP Biomedicals) for 45 seconds at a speed of 5.5 m/s. To remove cell debris and filter material, the tubes were centrifuged at 10,000 × g for 5 minutes. 600 µl of aqueous supernatant was transferred to a new 2 ml tube along extracted with one volume of chloroform. Tubes were centrifuged at 10,000 × g for 5 minutes, then the aqueous phase was kept. Purification and concentration of recovered DNA was performed by adding two volumes of MoBio solution C4 to the aqueous phase. This was passed over a MoBio spin filter. The spin filters were then washed with 400 µl of MoBio C5 solution. Residual C5 was removed by centrifuging the empty tubes at 10000 × g for two minutes. DNA was eluted by two 30 µl additions of MoBio C6 solution. Final eluted environmental DNA was stored in −80 °C freezer. DNA concentrations were determined using NanoDrop spectrophotometer.
9
Extraction and Quantification of Environmental DNA
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DNA extraction was performed using MoBio Powerlyzer Powersoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, USA). Samples that were immersed in solution were centrifuged at 13,000 × g for 5 min and the supernatant discarded. Samples were washed with 1 mL of cold 1× PBS (pH7.2) (Life Technologies, Melbourne, Australia) and centrifuged at 13,000 × g for 10 mins. DNA extraction was performed according to the manufacturer’s instructions with the following modifications. Samples were placed into bead tubes with solution C1 and heated at 65 °C for 10 min, prior to two cycles of bead beating at 6.5 m/s for 1 min using a FastPrep-24 bead beater (MP Biomedicals, Santa Ana, USA). Total DNA was eluted in 100 μL of sterile water. DNA concentration was quantified fluorometrically with a Qubit dsDNA HS Assay kit (Life Technologies).
10
Isolation and Analysis of Minichromosomes
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Minichromosomes were isolated essentially as described46 (link). Cells carrying minichromosome plasmids were grown in 1 L of SILAC media70 (link) (Kaiser SC-Arg-Lys-Trp (Formedium), supplemented with 20 mg/L heavy arginine-HCl (Arg10, CK Isotopes) and 30 mg/L lysine-2HCl (Lys6, CK Isotopes) or, for a control, 20 mg/L light arginine-HCl and 30 mg/L light lysine-2HCl) at 30 °C until log phase and harvested. After washing cells in ice-cold water supplemented with 2 mM PMSF, cells were resuspended in buffer H150 (25 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM MgCl2, 10% glycerol, 0.02% NP40, 1 mM PMSF, cOmplete Protease Inhibitor cocktail without EDTA (11873580001, Merck), PhosSTOP (4906845001, Merck), 5 mM nicotinamide) and disrupted using a FastPreP-24 bead beater (MP Biomedicals). Clarified lysates were prepared by centrifugation at 20,000 × g for 20 min three times. lacO-containing minichromosomes were isolated by immunoprecipitating LacI-3FLAG with anti-FLAG M2 antibody (12 μg, F1804, Merck) crosslinked to Dynabeads protein G (10765583, Fisher Scientific) by dimethyl pimelimidate. Proteins on purified minichromosomes were eluted in Elution buffer (50 mM ammonium bicarbonate, 0.1% RapiGest SF (186001860, Waters), 1 mM IPTG). The eluted proteins were analysed by SYPRO Ruby staining and western blot analysis.
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