For pigment extraction, R2A agar plates were inoculated evenly with 1 ml of a cell suspensions with a cell concentration of 107 CFU/ml. Plates were incubated for 24 h at 37°C. After incubation, biomass from two agar plates per strain was collected with sterile inoculation loops and transferred to sterile tubes with 2 ml H2O. 200 μl were used to determine the number of CFU/ml and the remaining suspensions were centrifuged for 10 min at 14,000 × g. Supernatants were discarded and pellets were frozen at −20°C. The frozen pellets were thawed at room temperature by adding 2 ml methanol (≥99.9%) and were transferred into Lysing Matrix B tubes (MP Biomedicals, Irvine, CA, United States). The samples were treated twice for 40 s in a high-speed benchtop homogenizer (FastPrep, MP Biomedicals, Irvine, CA, United States) at 6 m/s. Afterward, the samples were heated at 55°C for 5 min and centrifuged at 14.000 rpm for 10 min. The supernatant was removed and transferred to HPLC vials. For measuring the UV/Vis spectrum of the methanol extracts from the strains, 1 ml of the extracts was transferred into 10 mm UV-cuvettes (Brand, Wertheim, Germany). Measurement was performed using a UV/Vis spectrophotometer (NanoDrop 200C, Thermo Fisher Scientific Inc., Waltham, MA, United States) with methanol (≥99.9%) as a baseline.
Nanodrop 200c
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 200c is a compact and portable spectrophotometer designed for the measurement of small sample volumes. It provides accurate and reliable absorbance measurements for a wide range of applications, including nucleic acid and protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Fastprep, by MP Biomedicals (2 mentions)
Rnalater solution, by Qiagen (2 mentions)
4 bromanisole, by Molecular Research Center (2 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Zirconium silica beads, by Biospec (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Lysing matrix b tube, by MP Biomedicals (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Sw40ti rotor, by Beckman Coulter (1 mentions)
Optima l 80 xp, by Beckman Coulter (1 mentions)
Miseq platform, by Illumina (1 mentions)
Agencourt ampure beads, by Beckman Coulter (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Qubit rna high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
23 protocols using nanodrop 200c
1
Pigment Extraction and Quantification
2
Plasmid Standard Quantification by qRT-PCR
3
Transcriptional Response to Antagonist Peptide
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AC-25 clones were incubated with PP16 (without B cells) in either the presence or absence of antagonist peptide PG13 for 4 hours. Following incubation cells were flash frozen for microarray analysis. Total RNA was isolated using RNeasy Micro kits (Qiagen) and the quantity and quality confirmed using a NanoDrop 200 c (Thermo Fisher Scientific) and gel electrophoresis (Experion). Samples (50 ng) were amplified using Illumina TotalPrep RNA amplification kits (Ambion). Microarray analysis was conducted using 1.5 μg of biotinylated cRNA hybridized on Illumina Human WG-6 Expression Bead Chips (v2.0, Illumina) and then scanned on Illumina BeadStation 500GX. Raw data was collected with Illumina GenomeStudio software. Each hybridization was performed in triplicate. The data sets were pre-processed with quantile normalization, variance stabilization, and log2 transformation. Benjamini-Hochberg correction was employed to assess the occurrence of false positives by calculating the false discovery rate. Hierarchical clustering by Pearson’s correction and heatmap representations were generated using MultiExperiment Viewer v4.6.2 (Dana Farber Cancer Institute). Ingenuity Pathway Analysis v9 (IPA) (Ingenuity Systems) was used to select, annotate and visualize genes by function, network and canonical pathway.
4
Sucrose Density Gradient Ultracentrifugation
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The Ultra-clearTM centrifuge tube was first filled with 4 ml of 20% sucrose and then 4 ml of 30% sucrose buffer was carefully filled into the bottom of the tube using a needle-long syringe to keep the interface of the buffer as stable as possible. Sucrose buffer contains 2.38 g of HEPES, 10.17 g of MgCl2⋅6H2O, 200 g (20%) or 300 g (30%) of sucrose, and 0.15% DDM per liter of deionized water (pH 8.0). The pH was adjusted to 8.0 with sodium hydroxide solution. The tube was capped with parafilm and allowed to stand at room temperature for 2 h in a tilted state to form a sucrose density gradient. Thereafter, it was left for 1 h at 4°C. One milliliter of solubilized membrane protein (10 mg/ml) solubilized from the β-His strain was carefully overlaid on the sucrose density gradient. Ultracentrifugation was performed using OptimaTML-80XP and an SW40 Ti Rotor (Beckman coulter) at 40,000 rpm at 4°C for 16 h. After ultracentrifugation, 400 μl of the fraction was carefully separated from the upper layer of the tube, and A280 of each fraction was measured with NANO DROP 200c (Thermo Fisher Scientific). Fractions in which cytochrome oxidase activity was observed using TMPD were used for the next analysis.
5
Comprehensive Soil and Root Microbiome Analysis
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About 100 g of rhizospheric soil was mixed thoroughly and filtered with 2 mm sieve. The total DNA from soil samples (0.5 g) were extracted using FastDNA Spin Kit for Soil, according to manufacturer’s instructions. For DNA extraction from root endosphere, roots were surface sterilized according to Mukhtar et al. (2019) (link) and FastDNA Spin Kit specific for plant tissues was used. The quality of DNA was determined by using 0.9% agarose gel and quantity was measured by using Nanodrop (NanoDrop 200c Thermo Fisher Scientific, Waltham, MA, United States).
In total, 96 DNA samples; 12 rhizosphere and 12 root samples of each plant collected from the three geographic sites were used for amplification of 16S rRNA gene and Illumina (MiSeq) sequencing. The V3–V4 region of the 16S rRNA gene was amplified by using primers (Bakt_341F: CCTACGGGNGGCWGCAG and Bakt_805R: GACTACHVGGGTATCTAATCC), which were linked with unique identifier and adapter sequences (Supplementary Table 1 ). The detailed PCR conditions for amplicon sequencing were the same as described by Herlemann et al. (2011) (link). Amplified PCR products were purified with Agencourt AMPure beads (Beckman Coulter, Brea, CA, United States). Finally, about 10 ng of DNA from each sample was sequenced on the Illumina MiSeq platform by Macrogen (Geumcheon-gu, Seoul, South Korea).
In total, 96 DNA samples; 12 rhizosphere and 12 root samples of each plant collected from the three geographic sites were used for amplification of 16S rRNA gene and Illumina (MiSeq) sequencing. The V3–V4 region of the 16S rRNA gene was amplified by using primers (Bakt_341F: CCTACGGGNGGCWGCAG and Bakt_805R: GACTACHVGGGTATCTAATCC), which were linked with unique identifier and adapter sequences (
6
Tumor sample preparation and nucleic acid extraction
7
RNA Extraction and Sequencing from Plant Leaves
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Total RNA was isolated from mature leaves of differentially treated short cuttings using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). Three biological replicates were included per treatment. RNA size and quality were assessed by 1.2% agarose gel electrophoresis and spectrophotometer (NanoDrop 200c, Thermo Fisher Scientific), followed by the Agilent Bioananlyzer 2100 (Agilent Technologies, Santa Clara, CA, United States) and RNA Analysis Kits to assess whether the RNA has been degraded. After RNA quality verification, 9 cDNA libraries were constructed using a PCR-cDNA Sequencing Kit (SQK-PCS109) and then sequenced on the Nanopore PromethION plantform. Transcriptome analysis was performed by Biomarker Technologies Co., Ltd. (Beijing, China).
8
Purification of Brome Mosaic Virus Coat Protein
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Virions from brome mosaic virus (BMV) were produced in barley plants (Hordeum vulgare) and purified according to Nuñez‐Rivera et al.[48] The coat protein, free of RNA, was obtained as follows: BMV virions were dialyzed with a 14,000 Da membrane (Spectrum Laboratories) in a disassembly buffer (500 mM CaCl2, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 50 mM Tris‐HCl, pH 8) for 24 h at 4 °C. The dialyzed mixture was centrifuged at 147,000 g in a Beckman Coulter ultracentrifuge with a fixed angle 90 Ti rotor for 8.5 h at 4 °C. After centrifugation, aliquots (400 μL) were taken from the top to the bottom avoiding pellet resuspension. Each aliquot was analyzed in a UV‐vis spectrophotometer (Nanodrop 200c, Thermo Scientific), and the aliquots with A280≥0.1 and A280/A260≥1.5 were collected as pure coat protein (CP). The disassembled CP were dialyzed in a protein storage buffer (1 M NaCl, 1 mM EDTA, and 20 mM Tris HCl pH 7.2) for 12 h at 4 °C. The analysis by dynamic light scattering (DLS) showed the sole presence of the coat protein and do not showed any viral particle.
9
Caecum Tissue RNA Extraction Protocol
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Tissue samples from the caudal part of the caecum were cut into 20 mg pieces, placed immediately into RNA Later solution (Qiagen, UK), and stored at −70°C prior to RNA purification. Single tissue fragments were transferred into 1 ml of TRIzol Reagent (Molecular Research Center, USA), and homogenized with zirconium silica beads (BioSpec Products, USA) in a vortex mixer (Labnet, USA). To separate the phases, 50 μl of 4-bromanisole (Molecular Research Center, USA) was added. The whole content of the tube was centrifuged and the upper aqueous phase was collected for total RNA purification with the RNAeasy mini kit (Qiagen, UK), according to the manufacturer's instructions. Turbo DNA-free kit (Ambion, USA) was used for the treatment of RNA samples to remove genomic DNA. Both the purity and concentration of RNA were determined spectrophotometrically on NanoDrop 200c (Thermo Scientific, USA) and 1 μg of the total RNA immediately underwent reverse transcription with iScript cDNA Synthesis Kit (Bio-Rad, USA). The resulting cDNA was diluted 10-fold in UltraPure DNase/RNase-Free distilled water (Invitrogen, USA) and used as a template for real-time PCR, or stored at −20°C until used.
10
qRT-PCR Gene Expression Analysis
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