For gene silencing in whole femoral bone marrow, mice were sacrificed by CO2 asphyxiation, and femurs were harvested and immediately snap-frozen in liquid nitrogen. Frozen tissues were pulverized to form a powder using a SPEX 2010 Geno/Grinder (SPEX SamplePrep). Tissue lysates were prepared in Tissue and Cell Lysis Buffer (Epicentre) supplemented with 0.5 mg/mL Proteinase K (Epicentre). Tissue samples were mixed at 1400 RPM for 2 h at 65°C and centrifuged at 16,000 RCF to remove bone debris. mRNA levels in the supernatant were quantified using the QuantiGene 2.0 luminescent-based branched DNA (bDNA) assay kit and the QuantiGene 2.0 probes against Tie2 and GAPDH (Thermo Fisher Scientific) according to the manufacturer’s protocol. Luminescent signals were measured using a Tecan Infinite 200 PRO plate reader (Tecan). Standard curves for femur tissues and each target gene were constructed using samples from untreated mice to ensure optimal dilutions for assay samples that avoid luminescent signal saturation. Targeted gene silencing in treated mice was quantified by calculating the ratio of target gene luminescence to Gapdh gene luminescence, with all values normalized to target gene:Gapdh gene ratios from control mice.
Geno grinder 2010
Manufactured by SPEX
Sourced in United States, United Kingdom, Germany
The Geno/Grinder 2010 is a high-throughput sample preparation device designed for efficient homogenization and cell disruption of a wide range of sample types. It utilizes a unique grinding action to effectively break down samples into a fine, homogeneous powder or suspension, preparing them for subsequent analysis or processing.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Rneasy plant mini kit, by Qiagen (5 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Silwet l 77, by Merck Group (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Infinite 200 pro plate reader, by Tecan (2 mentions)
Proteinase k, by Illumina (2 mentions)
Tissue and cell lysis buffer, by Illumina (2 mentions)
Quantigene 2.0 luminescent based branched dna bdna assay kit, by Thermo Fisher Scientific (2 mentions)
Quantigene 2.0 probes against tie2 and gapdh, by Thermo Fisher Scientific (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
Direct zol rna miniprep, by Zymo Research (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Bluepippin, by Sage Science (2 mentions)
Direct zol rna miniprep kit with dnase 1 treatment, by Zymo Research (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
79 protocols using geno grinder 2010
1
Quantifying Gene Silencing in Femoral Bone Marrow
2
Quantifying Gene Silencing in Femoral Bone Marrow
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For gene silencing in whole femoral bone marrow, mice were sacrificed by CO2 asphyxiation, and femurs were harvested and immediately snap-frozen in liquid nitrogen. Frozen tissues were pulverized to form a powder using a SPEX 2010 Geno/Grinder (SPEX SamplePrep). Tissue lysates were prepared in Tissue and Cell Lysis Buffer (Epicentre) supplemented with 0.5 mg/mL Proteinase K (Epicentre). Tissue samples were mixed at 1400 RPM for 2 h at 65°C and centrifuged at 16,000 RCF to remove bone debris. mRNA levels in the supernatant were quantified using the QuantiGene 2.0 luminescent-based branched DNA (bDNA) assay kit and the QuantiGene 2.0 probes against Tie2 and GAPDH (Thermo Fisher Scientific) according to the manufacturer’s protocol. Luminescent signals were measured using a Tecan Infinite 200 PRO plate reader (Tecan). Standard curves for femur tissues and each target gene were constructed using samples from untreated mice to ensure optimal dilutions for assay samples that avoid luminescent signal saturation. Targeted gene silencing in treated mice was quantified by calculating the ratio of target gene luminescence to Gapdh gene luminescence, with all values normalized to target gene:Gapdh gene ratios from control mice.
3
Targeted Metabolomic Analysis of Pectoralis Major Muscle
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Around 3 g tissue samples of the pectoralis major muscle were cut out of the middle of the cranial region directly after bleeding. The samples were directly frozen in liquid nitrogen, shortly stored at −20°C, shipped on dry ice to Metabolon Inc. (Morrisville, NC), and finally stored at −80°C. The analysis was performed by Metabolon Inc. About 200 mg of the tissue samples were used for the analysis and mixed with 200 µL of a standard solution (carnosine-d4, anserine-d4, histidine-13C6, β-alanine-13C3, 15N) in 500 µL of acidified methanol containing 1% formic acid. The samples were homogenized by using glass beads and a tissue homogenizer (SPEX 2010 Geno/Grinder, SPEX SamplePrep). The samples were centrifuged, and the supernatant was used for liquid chromatography mass spectrometry/mass spectrometry (Agilent 1290/AB Sciex QTrap5500 LC MS/MS system, UHPLC BEH C18 column 2.1 × 100 mm 1.7 um). For separation, ion pair chromatography was used. The MS operated in positive mode by using electrospray ionization in MRM mode. The raw data were collected and processed by using the software Analyst 1.6.2 (SCIEX). Data were normalized to tissue weight. The used peak areas were measured against the areas of the used standards. A calibration curve, created by weighted least square regression, was used to determine the concentrations of the measured molecules.
4
Plant DNA Extraction and Quantification
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Frozen plant material was homogenized in a bead beater (2010-Geno/Grinder, SPEX SamplePrep®, USA) prior to DNA extraction using a Qiagen DNeasy kit according to the manufacturer's instructions. DNA samples were then quantified in a NanoDrop® 1000 Spectrophotometer (V 3.8.1, ThermoFisher Scientific Inc., Australia) and concentrations were standardized to 10 ng/µl for subsequent MSAP analyses.
5
Spray Inoculation of Pseudomonas syringae
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Spray inoculations were performed as previously described (79 (link)). P. syringae pv. tomato (Pto) DC3000 wildtype and COR- (defective in production of the phytotoxin coronatine) strains (80 (link)) were grown in overnight culture in King’s B medium supplemented with 50 μg/ml rifampicin, 50 μg/ml kanamycin, and 100 μg/ml spectinomycin and incubated at 28 °C. Cells were harvested by centrifugation and pellets resuspended in 10 mM MgCl2 to an A600 of 0.2, corresponding to 1 × 108 colony forming units (CFU)/ml. Immediately before spraying, Silwet L-77 (Sigma Aldrich) was added to a final concentration of 0.04% (v/v). Four-to five-week-old plants were uniformly sprayed with the suspension and covered with a clear plastic lid for 3 days. Three leaf discs (4-mm diameter) were taken using a biopsy puncher from three respective leaves of one plant and ground in collection microtubes, containing one glass bead (3-mm diameter) and 200 μl water, using a 2010 Geno/Grinder (SPEX) at 1500 rpm for 1.5 min. Ten microliters of serial dilutions from the extracts were plated on LB agar medium containing antibiotics and 25 μg/ml nystatin (Melford). Colonies were counted after incubation at 28 °C for 1.5 to 2 days.
6
Root RNA Extraction and cDNA Synthesis
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Materials detailed in Fig. 2a were sampled, snap frozen and stored at −80 °C for RNA extraction. The root tissue was ground to fine powder on 2010 Geno/Grinder® (SPEX SamplePrep) at 1200 RPM for 30 seconds, and RNA was extracted from the tissue powder by using Direct-Zol RNA MiniPrep (Zymo Research) according to the manufacturer’s protocol. Final elution was performed with 40 µL DNA/RNAase-Free water supplied with the kit and the eluted RNA was subsequently quantified using ND-1000 Spectrophotometer (NanoDrop Technologies). cDNA synthesis was then performed on 500 ng RNA by using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction in a 20 µL reaction and stored at −20 °C until use.
7
Microbiome 16S rRNA Sequencing Protocol
8
Genomic DNA Extraction and Sequencing
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Leaf tissue was collected using a hole punch from each accession in each cross once, and collected in duplicate from both sets of parents. Punched tissue was dried using a freeze dryer before being shipped to the AAFC Kentville Research and Development Centre in Kentville, Nova Scotia, Canada for DNA extraction. Tissue was lyophilized and then ground using a 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA). Whole-genome DNA was extracted using a NucleoSpin 96 Plant II kit (Machery-Nagel, Düren, Germany) with the following modifications to the kit protocol: samples were incubated in lysis buffer for 60 min, and were processed using a vacuum manifold with an additional step of filtering lysate through receiver plates before proceeding to the binding step. DNA samples were quantified using the QuantiFluor dsDNA System and the GloMax-Multi+Microplate Multimode Reader with Instinct (Promega, Madison, WI, USA).
Library preparation and DNA sequencing were performed at L’Institut de Biologie Intégrative et des Systèmes (IBIS) at Université Laval, Quebec City, Québec, Canada using an Illumina HiSeq 2000 and the GBS approach,26 (link) with the restriction enzyme ApeKI.34 (link) Each F1 progeny was sequenced once, while each parent in each cross was sequenced in duplicate as recommended by Gardner et al.35
Library preparation and DNA sequencing were performed at L’Institut de Biologie Intégrative et des Systèmes (IBIS) at Université Laval, Quebec City, Québec, Canada using an Illumina HiSeq 2000 and the GBS approach,26 (link) with the restriction enzyme ApeKI.34 (link) Each F1 progeny was sequenced once, while each parent in each cross was sequenced in duplicate as recommended by Gardner et al.35
9
Metabolite Extraction from Taxus Twig Samples
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For metabolite extraction, fresh twig samples from different Taxus species (25 mg each, n = 15) were transferred to 1.5-mL Eppendorf tubes, and 800 μL pre-cooled methanol/water (1:1, v/v) was added to the tube with two steel balls. All of the tubes were placed into a pre-cooled 48-well tube holder and ground using the 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA) for 2 min at a rate of 1900 strokes/min. The homogenized samples were extracted in 0.5-mL of the pre-cooled chloroform/methanol/water (v:v:v, 1:3:1) extraction solvent by vortexing for 15 min at 4 °C in the dark and then ultrasonication for 5 min on ice. The samples were centrifuged at 13,000 g for 15 min at 4 °C, and 550 μL of the supernatants were collected. The extracts were vacuum-dried and resuspended in a 50% methanol solution. The prepared extracts were then loaded onto the auto-sampler of the 2777C ultra-performance liquid chromatography (UPLC) system (Waters, Herts, UK) at 4 °C.
10
NMR-based Metabolomics Analysis of Rice
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For each of the three groups (i.e., WT, M, and ECE), 10 biological replicates of rice plants were used for the extraction and subsequent NMR-based metabolomics analysis. Each rice plant was extracted individually using a method modified from previous reports [23 (link),54 (link)]. Firstly, the rice sample-containing tube was snap-frozen in liquid nitrogen and the rice tissue was then swiftly ground into fine powder using a pre-cooled pestle. Pre-cooled methanol/water (v/v = 2/1, −20 °C) was added into the homogenized sample at a ratio of 600 µL per 100 mg powder. Afterwards, the mixture was further homogenized using a 2010 Geno/Grinder® (SPEX Sample Prep, Metuchen, NJ, USA) at 1300 rpm for 90 s. Then the homogenate mixture was sonicated in an ice bath with 10 cycles of 30 s sonication and 30 s break. Following centrifugation (14,489× g, 10 min, 4 °C), the supernatant was collected, and the remaining pellets were further treated twice using the same procedure. Three supernatants were combined and lyophilized after removal of methanol in vacuo (CentriVap Centrifugal Vacuum Concentrators, Labconco, MO, USA). Each dried extract was reconstituted into 600 μL phosphate buffer prepared as previously described. After a final centrifugation, 500 μL supernatant was transferred into a 5 mm NMR tube for NMR analysis.
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