Minimum essential medium-α (α-MEM) and penicillin/streptomycin (P/S) were obtained from Gibco (Gaithersburg, MD, USA). The CCK-8 assay was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Fetal bovine serum (FBS) was supplied by Atlas Biologicals (Fort Collins, CO, USA). Bicinchoninic acid (BCA) protein assay kit, β-glycerophosphate, ascorbic acid, dimethyl sulfoxide (DMSO) and amygdalin were obtained from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline was supplied by Welgene, Inc. (Daejeon, Republic of Korea). Anti-RUNX2, anti-BMP-2 and anti-SMAD1/5/9 antibodies were supplied by Abcam (Cambridge, UK). Anti-t-p38 and anti-p-p38 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were supplied by Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). PCR primers were supplied by Genotech (Daejeon, Republic of Korea). The SuperScript II Reverse transcription kit and SYBR-Green solution were purchased from Invitrogen (Carlsbad, CA, USA). Taq polymerase was purchased from Kapa Biosystems (Woburn, MA, USA). The OCN (cat. no: LS-F12230) and the OSN (cat. no: LS-F27540) ELISA kit was obtained from LSbio (Seattle, WA, USA).
Sybr green solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR green solution is a fluorescent dye used in molecular biology applications. It binds to double-stranded DNA, allowing for the detection and quantification of DNA in various procedures, such as real-time PCR and DNA gel electrophoresis. The solution provides a simple and sensitive method for DNA detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taq polymerase, by Roche (2 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)
Tmtp14250, by Merck Group (2 mentions)
Svgp01050, by Merck Group (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Epichemi 2 darkroom, by Analytik Jena (2 mentions)
Nd3 rna probe, by GenScript (2 mentions)
Mutant nd3 rna probe, by GenScript (2 mentions)
Amygdalin, by Merck Group (1 mentions)
α minimum essential medium α mem, by Thermo Fisher Scientific (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Dulbecco s phosphate buffered saline, by Welgene (1 mentions)
Superscript 2 reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca protein assay kit, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ai600 imager, by GE Healthcare (1 mentions)
Anti bmp2, by Abcam (1 mentions)
9 protocols using sybr green solution
1
Osteogenic Differentiation Protocol for Stem Cells
2
OmpR-His6 Binding to lcrGVsycD-yopBD Promoter
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Overproduction and purification of OmpR-His6 and its use in an EMSA to detect interactions of this recombinant protein with DNA sequences were performed as described previously [64 (link)]. Briefly, a 255-bp fragment of the promoter region of the lcrGVsycD-yopBD operon and a 304-bp 16S rDNA fragment as a nonspecific competitor were PCR-amplified using Y. enterocolitica genomic DNA as a template and the pairs of primers listed in Table S1 . The DNA fragments were incubated in OmpR-binding buffer (50 mM Tris-HCl pH 8.0, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 20 mM MgCl2, 12% glycerol, 100 µg/mL BSA, 0.1% Triton X-100) with increasing amounts of OmpR-His6 (molar ratio of 1:20–300) at 26 °C for 30 min. The samples were then loaded on a 4.2% native polyacrylamide gel (19:1 acrylamide/bisacrylamide, 0.2 × TBE) containing 2% glycerol and electrophoresis performed at 110 V for approximately 3 h. The gel was then stained in 1 × SYBRgreen solution (Invitrogen, Waltham, MA, USA) and visualized using a GE Healthcare AI600 imager (GE Healthcare, Chicago, IL, USA).
3
Comet Assay for DNA Damage
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Cells seeded in six-well plate (100,000 per well) were treated with typhoid toxin and incubated at 37 °C to permit DNA damage. Cells were harvested using a cell scraper in ice-cold PBS. Cells were combined 1:1 with 1.2% agarose (150 μl) and loaded onto 0.6% agarose (150 μl) immobilised on fully-frosted glass slides then topped with coverglass. Cells were lysed in lysis buffer (10 mM Tris pH10, 100 mM EDTA, 2.5 M NaCl, 1% Tx100. 1% DMSO) for 1 h, 4 °C. Agarose gel electrophoresis was performed for 25 min at 12 V in electrophoresis buffer (10 mM Tris pH10, 1 mM EDTA, 10 mM NaOH, 1% DMSO). DNA was labelled with SYBR green solution (Invitrogen, S7563). Comets were scored on a microscope with a 10x Plan Fluor objective on Nikon inverted microscope and analysed with software Comet Assay IV (Instem).
4
Osteoclastogenesis Inhibition via Rutin
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Minimum essential Eagle's medium, α-modification (α-MEM), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were supplied by Gibco; Thermo Fisher Scientific, Inc. Dulbecco's modified Eagle's medium (DMEM) was procured from Welgene, Inc. Alizarin Red S was obtained from Duksan Co., Ltd. RANKL was purchased from Peprotech, Inc. Rutin, dimethylsulfoxide (DMSO) and the TRAP assay kit were purchased from Sigma-Aldrich; Merck KGaA. Osteo strip well plates were purchased from Corning Inc. Anti-RUNX2, anti-BMP-2, anti-Ctsk and anti-MMP-9 antibodies were purchased from Abcam. Anti-phosphorylated (p)-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-p-c-Jun N-terminal kinase (JNK), anti-JNK, anti-p-p38, anti-p38, anti-NF-κB and anti-p-NF-κB antibodies were supplied by Cell Signaling Technology, Inc. Anti-NFATc1 was procured from BD Biosciences, and anti-c-Fos, anti-actin and anti-lamin B antibodies were purchased from Santa Cruz Biotechnology, Inc. Secondary antibodies were procured from Jackson ImmunoResearch Laboratories, Inc. The reverse transcriptase kit and SYBR-Green solution was supplied by Invitrogen; Thermo Fisher Scientific, Inc. Taq polymerase was purchased from Kapa Biosystems; Roche Diagnostics. PCR primers were purchased from Genotech Corp. All of the chemicals used in the experiments were analytical grade for cell culture.
5
Limnological Sampling of Lake Biwa
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Water samples were collected monthly from May 2018 to April 2019 at a pelagic station (water depth, ca. 73 m) on Lake Biwa (35°13′09.5″N, 135°59′44.7″E) from two water depths, representing the epilimnion (5 m) and the hypolimnion (65 m) (24 samples in total). Vertical profiles of chlorophyll a concentration, temperature, and dissolved oxygen were collected using a RINKO CTD profiler (ASTD102; JFE Advantech). The collected lake water was immediately sequentially filtered through a 200-μm mesh, 5-μm polycarbonate filter (TMTP14250; Merck Millipore) and a 0.22-μm-pore-size Sterivex cartridge (SVGP01050; Merck Millipore), using a peristaltic pump system onboard. Filtration was performed until the Sterivex cartridge was clogged (1 to 2.5 L of lake water was filtered for each cartridge), and at least four Sterivex cartridges were collected for each sample. The filters were flash-frozen in a dry ice-ethanol bath, transported to the laboratory on dry ice, and stored at −80°C until further processing. Water samples were collected between 8:00 a.m. and 11:00 a.m. on each sampling day and processed to the freezing step within 1 h after collection. Prokaryotic cell abundance was determined for each sample using a flow cytometer (CytoFLEX; Beckman Coulter) following fixation of the water sample with 1% glutaraldehyde and staining with 0.25× SYBR green solution (S7563; Invitrogen).
6
Quantitative Real-Time qRT-PCR Primer Design
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The Primer3plus web tool was used to design quantitative real time qRT-PCR primers. All primer sequences are displayed as Supplementary data in Table S2. Primers were validated with a standard curve based on a serial dilution of cDNA to determine primer annealing efficiency. Each qRT-PCR reaction was performed in duplicate and contained 5 µl SYBR green solution (Invitrogen), 0.5 µl of forward and reverse primer (10mM) (Sigma), 2 µl milliQ water and 4 µl of cDNA. The PCR reaction was performed in a 96 well plate and analyzed by the StepOne System (ABI Prism, Applied Biosystems). Relative expression levels were calculated using the delta delta Ct method (Livak and Schmittgen, 2001) . To correct for sample variation, expression was normalized against the geometric mean of two stably expressed reference genes (Vandesompele et al., 2002) . Expression in CPB was normalized against Ld_arf1 and Ld_rp4 (Shi et al., 2013) (link), expression in S. gregaria was normalized against and Sg_gapdh (Van Hiel et al., 2009) .
7
Seasonal Monitoring of Microbial Community in Lake Biwa
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Water samples were collected monthly from May 2018 to April 2019 at a pelagic station (water depth ca. 73 m) on Lake Biwa (35°13′09.5″ N, 135°59′44.7″ E) from two water depths, representing the epilimnion (5 m) and hypolimnion (65 m) (24 samples in total). Vertical profiles of chlorophyll-a concentration, temperature, and dissolved oxygen were collected using a RINKO CTD profiler (ASTD102; JFE Advantech). The collected lake water was immediately sequentially filtered through a 200 µm mesh, 5 µm polycarbonate filter (TMTP14250; Merck Millipore), and 0.22 µm pore Sterivex cartridge (SVGP01050; Merck Millipore), using a peristaltic pump system onboard. Filtration was performed until the Sterivex cartridge was clogged (1-2.5 liters of lake water were filtered for each cartridge), and at least four Sterivex cartridges were collected for each sample. The filters were flashfrozen in a dry-ice ethanol bath, transported to the laboratory on dry ice, and stored at -80°C until further processing. Water samples were collected between 8:00 am and 11:00 am on each sampling day and processed to the freezing step within 1 h after collection. Prokaryotic cell abundance was determined for each sample using a flow cytometer (CytoFLEX; Beckman Coulter) following fixation of the water sample with 1% glutaraldehyde and staining with 0.25× SYBR Green solution (S7563; Invitrogen).
8
RNA-Protein Binding Assay with ND3 Probes
9
RNA-Protein Binding Assay with ND3 Probes
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ND3 RNA probe (5′-UCCACCCCUUACGAGUGCGGCUUCGACCCUAUAUC-3′) and mutant ND3 RNA probe (5′-UCCACCCCUUACAAAUAAAGCUUCGACCCUAUAUC-3′) were purchased from GenScript (Piscataway, NJ). RNA-protein binding was performed in 20 μl reactions containing 1μM RNA probe. The binding conditions were 10 mM Tris at pH 7.5, 10 mM HEPES at pH 7.5, 20 mM KCl, 2 mM MgCl2, 1.5 mM DTT, 5% glycerol. After incubation at RT for 30 min, samples were run at 100 V on a pre-electrophoresed 6% polyacrylamide gel containing 0.5×TBE for 1 hour in ice water bath. Gel was stained by SYBR Green solution (Life Technologies, Grand Island, NY) for 20 min at RT. Then stained gel was rinsed with water for three times and visualized by EpiChemi II Darkroom (UVP, Upland, CA)
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