RNA samples were prepared from three independent SCG axons and parental soma. Samples were prepared according to Affymetrix protocols (Affymetrix). RNA quality and quantity was monitored using the Bioanalyzer (Agilent) and NanoDrop (ThermoFisher Scientific) respectively. Per RNA labeling, 200 nanograms of total RNA was used in conjunction with the Affymetrix recommended protocol for the GeneChip 2.0 ST chips. The hybridization cocktail containing the fragmented and labeled cDNAs was hybridized to the Affymetrix Rat Genome 2.0 ST GeneChip. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA). An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using Affymetrix AGCC software. Partek Genomic Suite was used to normalize for Robust Multi-array Average (Robust Multichip Analysis), summarize the probes, log2 transform the data and conduct ANOVA analysis.
Biotinylated anti streptavidin
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated anti-streptavidin is a laboratory reagent used to detect and bind to streptavidin, a protein commonly used in various bioassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Genechip scanner 3000, by Thermo Fisher Scientific (7 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Streptavidin phycoerythrin solution, by Thermo Fisher Scientific (6 mentions)
Bioanalyzer, by Agilent Technologies (6 mentions)
Fluidics station, by Thermo Fisher Scientific (4 mentions)
Agcc software, by Thermo Fisher Scientific (2 mentions)
Cytoscan hd genechip, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Streptavidin cy3, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
D3159, by Merck Group (1 mentions)
Image pro, by Media Cybernetics (1 mentions)
Streptavidin fitc, by Vector Laboratories (1 mentions)
Dapi anti fade solution, by Vector Laboratories (1 mentions)
Rnase a, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
Bioprime dna labeling system, by Thermo Fisher Scientific (1 mentions)
Nucleobond pc100 kit, by Macherey-Nagel (1 mentions)
Cot 1 dna, by Roche (1 mentions)
Biotin 16 dutp, by Roche (1 mentions)
15 protocols using biotinylated anti streptavidin
1
Rat Genome 2.0 ST GeneChip Analysis
2
FISH with Biotinylated Oligo-Probes
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FISH with single-stranded oligo-probes was carried out according to the protocol [36 (link)] with several modifications. The probes were synthesized with both 3’ and 5’ ends labeled by biotin. Slides with metaphase spreads were treated in RNAse (Sigma-Aldrich, R6513, Merk KGaA, Darmstadt, Germany) stock solution (10 µg/mL) diluted 1:200 with 2xSSC for 45–60 min at 37 °C and washed 3 times for 5 min with 2xSSC at RT. Metaphase spreads were denatured in solution (70% formamide, 2xSSC) for 3–5 min at 72 °C and dehydrated in an ethanol series at −20 °C. Then, slides were incubated in the hybridization mixture: biotinylated oligo-probe in Hybrisol (Molecular Probes, Eugene, OR, USA) for 16–18 h at 37 °C. After post-hybridization washing, the slides were incubated with streptavidin conjugated with Alexa 546 (ThermoFisher Scientific, Waltham, MA, USA). Biotinylated antistreptavidin (Vector Laboratories, Burlingame, CA, USA) was used to amplify the signal, and then again streptavidin conjugated with Alexa 546 was used; all concentrations corresponded to the protocol of the manufacturer. The slides were finally mounted in Prolong Gold Antifade with DAPI (ThermoFisher Scientific, Waltham, MA, USA) and stored refrigerated in the dark.
3
Biotin-labeled DNA Hybridization Assay
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The method was according to Zhou et al. (2013) with minor modifications. Genomic DNA of Oriental ‘Sorbonne’ and L. regale was isolated using the CTAB method (Rogers and Bendich 1988 ), and labeled with biotin-16-dUTP as the probe, according to the manufacturer’s instructions (Biotin- Nick translation Mix 11745824910; Roche, Mannheim, Germany). The hybridization mix (40 μL) contained 50% deionized formamide, 10% dextran sulfate, 2× SSC (0.3 M NaCl plus 30 mM sodium citrate, pH 7.0), 0.25% sodium dodecyl sulfate (SDS), 25–50 ng probe DNA and 2 μg herring sperm DNA (D3159, Sigma-Aldrich). Signal was detected with Streptavidin-CY3 (Invitrogen, Camarillo, CA) and Biotinylated anti-Streptavidin (Vector Laboratories, Burlingame, CA). After counterstaining with DAPI (Roche), the slides were observed under a fluorescence microscope (BH41; Olympus). Images were taken with an attached CCD (Micropublisher 3.3 RTV; QImaging, Surrey, Canada) driven by Image-Pro® (Media Cybernetics, Rockville, MD).
4
Affymetrix Microarray Gene Expression Protocol
5
Whole-Genome Microarray Analysis Protocol
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Samples were prepared according to Affymetrix protocols (Affymetrix, Inc). DNA quality and quantity was ensured using Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc) respectively. Per DNA labeling, 200 nanograms of genomic DNA were used in conjunction with the Affymetrix recommended protocol for CytoScan HD array kit and reagents (catalog# 901835). The hybridization cocktail containing the fragmented and labeled DNAs was hybridized to The Affymetrix CytoScan HD GeneChip. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA). An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. The .Cel files were generated from the scanned images using Affymetrix AGCC software and the .cyhd.cychp files were generated by the Chromosome Analysis Suite (ChAS) Version 2.1 software.
All the analyses were done with ChAS default parameters for LOH and Copy Number State (CNS). A description of the samples used for this analysis is listed inSupplementary Table 5 .
All the analyses were done with ChAS default parameters for LOH and Copy Number State (CNS). A description of the samples used for this analysis is listed in
6
Affymetrix Microarray Gene Expression Analysis
7
Fluorescent in situ Hybridization for rDNA Mapping
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FISH for localizing major rDNA was performed as described previously [49 (link)] with some modifications. Chromosome preparations were first treated with RNase A (200 ng/μL) in 2× SSC for 1 h followed by two washes in 2× SSC for 5 min and 30 min incubation in 5× Denhardt’s solution, all at 37 °C. Chromosomes were denatured in 70% formamide in 2× SSC for 3.5 min at 68 °C. The probe mixture for one slide contained 40 ng of biotin-labeled 18S rDNA probe and 25 μg of sonicated salmon sperm DNA in 10 µL of 50% deionized formamide, 10% dextran sulfate in 2× SSC. Hybridization signals were visualized and amplified by three-step detection, Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc., West Grove, PA, USA), biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA, USA) and Cy3-conjugated streptavidin. The preparations were counterstained with 0.5 µg/mL DAPI in DABCO-based antifade.
8
Transcriptome Profiling using Affymetrix Mouse GeneChip
9
Fluorescence In Situ Hybridization Protocol
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UM-SCC1 cell culture slides were fixed using methanol/acetic acid (3:1) and pretreated with RNase A and pepsin [Sigma-Aldrich, Munich, Germany]. Next, the sections were rinsed in PBS at room temperature, followed by 4% paraformaldehyde/PBS, dehydrated, and air-dried. Bacterial artificial chromosome clones (BAC) for SEC62 (RP11-379K17) and SOX2 (RP11-203N24) purchased from ImaGenes (Berlin, Germany) were extracted using the NucleoBond® PC100 Kit (Macherey-Nagel, Dueren, Germany). BAC clones were labeled by BioPrime® DNA Labeling System (Invitrogen™, Life Technologies, Darmstadt, Germany) and a centromeric probe for chromosome 10 (D10Z3) as an internal hybridization control was labeled with digoxigenin by nick translation (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer´s instructions. Dual color hybridization was performed in 50% formamide/2× SSC and COT-1 DNA (Roche Diagnostics) at 37°C overnight. Stringency washes were performed three times in 50 % formamide/2 × SSC at 42°C and two times in 2× SSC at 42°C. Immunofluorescence detection of the biotin signals was performed using Streptavidin-FITC and biotinylated anti-Streptavidin [Vector Laboratories, Burlingame, CA] for digoxigenin using anti-dig-Cy3 (Roche Diagnostics GmbH, Mannheim, Germany), and the nuclei were counterstained with a DAPI anti-fade solution (Vector Laboratories, Burlingame, CA, USA).
10
Cytogenetic Analysis of Festulolium Hybrids
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FISH using a telomere probe, GISH, and image analysis were performed according to Akiyama et al. (2010) with slight modifications. Genomic DNA of L. multiflorum and F. arundinacea was directly labeled with fluorescein-12-dUTP (PerkinElmer Co., Billerica, MA, USA) and Texas Red-5-dUTP (PerkinElmer Co.), respectively, by a nick translation kit (Roche, Basel, Switzerland). For the telomere probe, Arabidopsis telomere repeat sequences (Richards and Ausubel 1988 (link)) were PCR labeled with biotin-16-dUTP (Roche) and detected by Streptavidin DyLight 549 (Vector Laboratories, Burlingame, CA, USA) as the first layer of signal amplification, biotinylated anti-streptavidin (Vector Laboratories) as the second layer and again with Streptavidin DyLight 549 as the third layer. Chromosome spreads were observed under an Olympus BX61 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a DP72 CCD camera (Olympus Optical Co., Ltd.). Based on the GISH results, the ratio of the Festuca-specific genomic component to the total festulolium genome (referred to as the f ratio) was analyzed using intensity values in DAPI images by Adobe Photoshop CS6 Extended (Adobe Systems Inc., San Jose, CA, USA) on a Windows 7 platform (Microsoft Corporation, Redmond, WA, USA). At least 6 chromosome spreads were analyzed to determine the f ratio in each sample.
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