All substitutions or indels identified through WES were confirmed by Sanger sequencing, and available family members were sequenced for segregation analysis (Supplementary Figs. S1 –S7 ). Potential de novo variants were confirmed by sequencing of parental DNA and excluding nonpaternity. Polymerase chain reaction (PCR) of CYP1B1 and FOXC1 was performed on Veriti 96 Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA). The protocol used for CYP1B1 amplification has previously been published.15 (link) PCR for FOXC1 was performed according to PCR Phusion High-Fidelity DNA Polymerase Protocol (Thermofisher Scientific, Waltham, MA, USA) using the GC-buffer and 2x S-Solution (Solis BioDyne, Tartu, Estonia). Big Dye Terminator Cycle Sequencing Kit version 1.1/3.1 (Thermofisher Scientific) was used for the Sanger reactions and a 3130xl Genetic Analyzer (Applied Biosystems) performed capillary sequencing. Sequences were visualized by Chromas version 2.6.6 (Technelysium, Brisbane, Australia). Primers are available on request. Multiplex ligation-dependent probe amplification (MLPA) was used to confirm FOXC1 CNVs using the SALSA MLPA P054-B2 FOXL2-TWIST1 probemix (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions. MLPA amplicons were analyzed using a 3130xl Genetic Analyzer (Applied Biosystems) and Sequence Pilot version 5.0 (JSI medical systems).
3130xl genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Australia, Japan, United Kingdom, Netherlands, France, Canada, Switzerland, Spain
The 3130xl Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing and fragment analysis. It features 16 capillaries and a laser-induced fluorescence detection system. The 3130xl enables high-throughput genetic analysis with its automated sample handling and data processing capabilities.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (104 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (23 mentions)
Hi di formamide, by Thermo Fisher Scientific (21 mentions)
Exosap it, by Thermo Fisher Scientific (19 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (18 mentions)
Qiaquick gel extraction kit, by Qiagen (17 mentions)
Qiaquick pcr purification kit, by Qiagen (15 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (14 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (13 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (13 mentions)
Genemapper software, by Thermo Fisher Scientific (12 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (12 mentions)
Genemapper 4, by Thermo Fisher Scientific (10 mentions)
Pgem t easy vector, by Promega (8 mentions)
Qiaamp dna mini kit, by Qiagen (8 mentions)
Genemapper v4, by Thermo Fisher Scientific (8 mentions)
Lightcycler 480, by Roche (7 mentions)
Seqscape software v2, by Thermo Fisher Scientific (7 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (7 mentions)
Dneasy blood tissue kit, by Qiagen (7 mentions)
645 protocols using 3130xl genetic analyzer
1
Confirming Genetic Variants through Sanger Sequencing
2
Cloning and Sequencing of AOx Gene
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Each PCR fragments were subsequently cloned into TA cloning vector (pGEM-T Easy, Promega, USA) and individual DNA sequencing was performed (3130xl Genetic Analyzer, Applied Biosystems). BglII and NdeI as unique restriction enzyme sites present in the overlapping region of both the PCR fragments and in the pGEMT vector backbone, respectively, were chosen for checking the proper orientation of the cloned fragments. First PCR product harboring the start codon of AOx open reading frame was ligated with second PCR fragment harboring the stop codon, at the common restriction site, thus confirming the full length functional clone of AOx from A.terreus MTCC6324. The full length clone of AOx in TA vector was confirmed through primer walking of both DNA strands (3130xl Genetic Analyzer, Applied Biosystems).
3
Comprehensive Genomic Profiling of Cell Lines
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Cell line DNA sequence was determined by sequencing with BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Samples were electrophoresed on a 3130xl Genetic Analyzer (Applied Biosystems) and sequence analysis was performed using the DNA sequencing software Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA). Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed using the SALSA MLPA probemix P056-B1 TP53 (MRC-Holland, Amsterdam, the Netherlands) according to manufacturer's instructions. Samples were electrophoresed on a 3130xl Genetic Analyzer (Applied Biosystems) and MLPA analysis was performed using the genotype analysis software Genemarker (SoftGenetics, State College, PA, USA). Furthermore, whole exome sequencing data from Woodward et al. [5] were reanalysed regarding TP53 mutational status using ANNOVAR.
Targeted analysis for the BRAF V600E mutation was done by direct sequencing as described previously [6] .
Targeted analysis for the BRAF V600E mutation was done by direct sequencing as described previously [6] .
4
Fluorescent DNA Glycosylase Cleavage Assay
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The sequences of the fluorescently labeled oligodeoxynucleotide substrates are shown in Figure 1A and prepared as previously described (12 (link)). DNA glycosylase cleavage assays for E. coli MUG and UNG were performed at 37°C for 60 min in a 10-μl reaction mixture containing 10 nM DNA substrate, 100 nM DNA glycosylase, 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM EDTA and 2 mM 2-mercaptoethanol. The glycosylase assay for Tth UDGb was performed as previously described (16 (link)). The resulting abasic sites were cleaved by incubation at 95°C for 5 min after adding 0.5 μl of 1 N NaOH. Reactions were quenched by addition of an equal volume of GeneScan stop buffer. Samples (3.5 μl) were loaded onto a 7 M urea-10% denaturing polyacrylamide gel. Electrophoresis was conducted using an Applied Biosystems 3130xl Genetic analyzer. Cleavage products and remaining substrates were quantified using GeneMapper analysis software for Applied Biosystems 3130xl Genetic analyzer.
5
Genetic Diversity Analysis of Cherry Cultivars
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The 230 cultivars were genotyped for 31 nuclear SSRs [26 (link)–28 (link)] (S2 Table ) and 5 chloroplast SSRs (cpSSRs) (Cmcs1–3, Cmcs5, and Cmcs7) [29 ]. PCR amplification was performed in a 10-μL solution containing 5 μL of 2× Green GoTaq G2 Hot Start Master Mix (0.4 mM each dNTP, Taq DNA polymerase, and 4 mM MgCl2, pH 8.5; Promega, Madison, WI, USA), 20 pmol of each forward primer labeled with a fluorescent dye (5-FAM or 5-HEX) and unlabeled reverse primer, and 2.5 ng of genomic DNA. Amplification was performed in 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. PCR products were separated and detected with a 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). The size of each amplified band was determined by comparison with a set of internal-standard DNA fragments (400HD ROX, Life Technologies) in GeneMapper software v. 5.0 (Life Technologies).
6
Characterization of Glioblastoma Cell Lines
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The human glioblastoma cell lines U251-MG (mutant p53; expressing R273H mutation) and U87-MG (wild-type p53) were a generous gift of Dr. Gad Lavie (Sheba Medical Center) while normal human astrocytes (NHA) were received as a gift of Prof. Chaya Brodie (Bar-Ilan University). The cell lines have been tested and authenticated before the experiments were established by the Promega PowerPlex 16 HS and analyzed using the 3130xl Genetic Analyzer (Life Technologies) and GeneMapper IDX software U251-MG and U87-MG were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Normal human astrocytes (NHA) served as controls and were cultured in Astrocyte Basal Medium (ABM; Lonza) supplemented with 0.1% rhEGF, 0.25% insulin, 0.25% ascorbic acid, 0.1% GA-1000, 1% L-glutamine, 3% FBS and 1% penicillin/streptomycin. All the cell lines were cultured at 37°C, in a humidified 5% CO2 atmosphere.
7
PCR-based Sequencing of Sodium Channel Genes
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Partial scn4aa, scn4ab, scn1b, scn2b and scn4b sequences were obtained using PCR primers (S1 Table ). PCR was performed in a Biorad Peltier thermal cycler (Biorad Laboratories, Hercules, CA, USA) using Dreamtaq polymerase (Thermo Fisher Scientific Inc.). The cycling conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min and a final extension of 72°C for 10 min. PCR products were separated by electrophoresis in 1% agarose gel. Bands of predicted molecular masses were excised and purified using FavorPrep™ Gel Purification Mini Kit (Favorgen Biotech Corp., Ping-Tung, Taiwan) according to manufacturer’s protocol. Purified PCR products were subjected to cycle sequencing using BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, California) and sequenced using the 3130XL Genetic Analyzer (Life Technologies Corporation).
8
Phylogenetic Analysis of PRRSV Samples
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Representative PRRSV positive samples were further typed using the corresponding ORF5 gene modified methods [19 (link),20 (link)]. Another protocol was applied for the real-time RT-PCR NA positive samples from Asia [3 (link),4 (link),21 (link)]. The corresponding ORF5 PCR bands of the expected sizes were excised from the agarose gel and recovered using the QIAquick® Gel Extraction Kit (Qiagen). Sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies) on the 3130xl Genetic Analyzer (Life Technologies). An similarity-based tree was constructed for phylogenetical analysis based on 628 or 611 nucleotides for EU and NA ORF5 regions, respectively, using the UPGMA algorithm with BioNumerics software (version 5.1; Applied Maths, Sint-Martens-Latem, Belgium).
9
Screening for KIF11 Mutations
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To screen for KIF11 mutations, the 22 exons and their corresponding splice-sites were amplified, as described [6 (link)]. Purity and size of amplicons were evaluated by agarose gel electrophoresis using GeneRuler™ 100 bp (Thermo Scientific) DNA size ladder. Direct sequencing was performed on a 3130xl Genetic Analyzer (Life Technologies). All sequences were analyzed with CLC Main Workbench 6© (CLC Bio), using reference sequence NG_032580.
10
Acrylamide Gel Electrophoresis of LM-PCR
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The LM-PCR mixture was ethanol precipitated after thermocycling and resuspended in 5 μl of 10 mM Tris-HCl + 0.1 mM EDTA. As gel matrix, 10–16% acrylamide/bis-acrylamide (19:1; Carl Roth) in a Protean II xi Cell was used. Gels were stained for 60 min with 3× GelRed solution (Biotum) and imaged with a gel documentation system. For high-resolution fragment analysis, the PCR was directly analyzed by a 3130xl Genetic Analyzer (Life Technologies GmbH) using a POP7 36-cm capillary and Internal Lane Standard 600 (Promega GmbH, Mannheim, Germany). The chromatograms were analyzed and visualized as virtual gel images by custom-built MATLAB (MathWorks, Natick, MA) routines.
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