Abi prism 3500xl

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ABI PRISM 3500xl is a genetic analyzer designed for DNA sequencing and fragment analysis. It uses capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments. The system provides high-throughput performance and can process multiple samples simultaneously.

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Lab products found in correlation

Genescan 600 liz, by Thermo Fisher Scientific (2 mentions) Qiaamp dna mini kit, by Qiagen (2 mentions) Improm 2 reverse transcriptase, by Promega (2 mentions) Abi prism 3130xl, by Thermo Fisher Scientific (1 mentions) Abi prism bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Nucleon bacc3 kit, by Cytiva (1 mentions) Qiaamp dna blood mini kit, by Qiagen (1 mentions) Genemarker 1, by SoftGenetics (1 mentions)

14 protocols using abi prism 3500xl

IDUA Gene Sequencing Protocol

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DNA was extracted following the manufacturer’s protocol with the DIAtomt DNA Prep100 kit (Isogene Lab. Ltd., Russia).
The 14 exons and exon–intron boundaries of the IDUA gene were amplified from DNA samples. Primers and PCR reaction conditions have been previously described (Beesley et al., 2001 (link)). Sanger sequencing of each one of the 14 exons was performed according to the manufacturer’s protocol on an ABI Prism 3500XL (Applied Biosystems). PCR products containing mutations were re-sequenced in both directions. The mutations were further confirmed where possible by restriction analysis (data not shown).

Voskoboeva E.Y., Bookina T.M., Semyachkina A.N., Mikhaylova S.V., Vashakmadze N.D., Baydakova G.V., Zakharova E.Y, & Kutsev S.I. (2022). Mucopolysaccharidosis Type I in the Russian Federation and Other Republics of the Former Soviet Union: Molecular Genetic Analysis and Epidemiology. Frontiers in Molecular Biosciences, 8, 783644.

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ARSB Gene Mutation Screening Protocol

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DNA extraction was carried out according to the manufacturer’s protocol using a DIAtomt DNA Prep100 kit (Isogene Lab. Ltd., Russia).
The eight exons and exon–intron boundaries of the ARSB gene were amplified from DNA samples. Primers and PCR reaction conditions have been previously described (Petry et al., 2003 (link)). Sequencing was performed according to the manufacturer’s protocol on an ABI Prism 3500XL (Applied Biosystems).
Blood specimens (unidentified) were provided by the neonatal screening center in Makhachkala (Republic of Dagestan). PCR-RFLP analysis using PspN4 I (GGN^∧NCC) restriction endonuclease (SibEnzyme, Russia) was developed for the detection of NM_000046.5:c.194C>T mutation. To estimate the frequency of the NM_000046.5:c.194C>T mutation, 500 DNA samples were examined.
The frequency of the disorder was calculated from the Hardy–Weinberg equation: p²+ 2pq + q² = 1. The confidence interval for frequencies was calculated by the Wilson method.

Voskoboeva E., Semyachkina A., Miklyaev O., Gamzatova A., Mikhaylova S., Vashakmadze N., Baydakova G., Omzar O., Pichkur N., Zakharova E, & Kutsev S. (2022). Epidemiology and Genetics of Mucopolysaccharidosis Type VI in Russia. Frontiers in Molecular Biosciences, 8, 780184.

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Sanger Sequencing of G6PD A- Variants

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All G6PD A− positive PCR products were confirmed with Sanger nucleotide sequencing. After purification with the NucleoSpin^® Gel and PCR clean-up kit (Macherey–Nagel, Duren, Germany), samples resuspended in HiDi™ formamide were sequenced using an ABI PRISM BigDye^® terminator V3.1 cycle sequencing kit and processed on ABI PRISM 3130xl and ABI PRISM 3500xl genetic analyzers (Applied Biosystems™/Life Technologies, Carlsbad, USA). BLAST queries were conducted for preliminary identifications of the sequences and thereafter analysed with MAFFT v 7.182 and CLC sequence viewer software.

Awandu S.S., Raman J., Makhanthisa T.I., Kruger P., Frean J., Bousema T., Niemand J, & Birkholtz L.M. (2018). Understanding human genetic factors influencing primaquine safety and efficacy to guide primaquine roll-out in a pre-elimination setting in southern Africa. Malaria Journal, 17, 120.

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Characterizing CTX-M-1 β-lactamase Genes

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Sequencing was performed from the PCR product amplified for ISEcp (Zurita et al., 2016 (link)). When negative for this insertion sequence, the amplified product for the gene using bla_CTX–M–1 (Fei Tian et al., 2011 (link); Cantón et al., 2012 (link); Dierikx et al., 2013 (link)) was characterized for bidirectional Sanger sequencing on ABI-PRISM 3500 XL (Applied Biosystems) following the manufacturer’s recommendations. For the interpretation and alignment of the sequencing results, Chromas was used to evaluate the electropherogram, and ClustalW was used to achieve alignment of the sequences. Using BLAST, the sequences were compared with the NCBI database.

Menck-Costa M.F., Baptista A.A., Gazal L.E., Justino L., Sanches M.S., de Souza M., Nishio E.K., Queiroz dos Santos B., Cruz V.D., Berbert J.V., Gonçalves B.C., Andrade G., Vespero E.C., Nakazato G, & Kobayashi R.K. (2022). High-Frequency Detection of fosA3 and blaCTX–M–55 Genes in Escherichia coli From Longitudinal Monitoring in Broiler Chicken Farms. Frontiers in Microbiology, 13, 846116.

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Mutagenic OPA1 Variants Generation

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The pCCEY plasmid containing human OPA1 isoform 1 WT was kindly donated by Guy Lenaers. Mutagenic OPA1 variants were designed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and following the manufacturer´s protocol. The actual generation of specific mutations was confirmed by sequencing using the ABI PRISM 3500 xl Applied Biosystems (FONDEQUIP EQM150077) at Pontificia Universidad Católica de Chile or sequenced at Macrogene inc., Seoul, South Korea. pCCEY plasmid encoding GTPase mutant OPA1 c.899G>A (G300E) and GED mutant OPA1 c.2708delTTAG was kindly donated by Guy Lenaers and used in previous studies (Olichon et al., 2007b (link); Eisner et al., 2014 (link)). For primers’ details, see Table 1.

Cartes-Saavedra B., Macuada J., Lagos D., Arancibia D., Andrés M.E., Yu-Wai-Man P., Hajnóczky G, & Eisner V. (2022). OPA1 Modulates Mitochondrial Ca2+ Uptake Through ER-Mitochondria Coupling. Frontiers in Cell and Developmental Biology, 9, 774108.

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Genomic DNA Extraction and GBA1 Gene Analysis

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Molecular analysis: Genomic DNA was extracted from peripheral blood leukocytes, cultured fibroblasts and lymphoblasts using QIAamp DNA blood Mini Kit (Qiagen GmbH, Hilden, Germany) or Nucleon BACC3 kit (Amersham Biosciences, Buckinghamshire, UK). GBA1 gene exons and most intronic regions were PCR amplified using primers designed by reference to the genomic sequence (GenBank J03059.1) to selectively amplify the gene and not the homologous pseudogene (GenBank J03060.1) as previously described [13 (link)].
PCR products were analyzed by automated sequencing (ABI Prism 3500xl genetic analyzer, Applied Biosystems, Foster City, CA, USA). Putative mutations were confirmed by sequencing duplicate PCR products and by the DNA analysis from parents.

Ciana G., Dardis A., Pavan E., Da Riol R.M., Biasizzo J., Ferino D., Zanatta M., Boni A., Antonini L., Crichiutti G, & Bembi B. (2020). In vitro and in vivo effects of Ambroxol chaperone therapy in two Italian patients affected by neuronopathic Gaucher disease and epilepsy. Molecular Genetics and Metabolism Reports, 25, 100678.

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Detection and Characterization of ESBL and AmpC Resistance Genes

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A previously described PCR method was used for the detection of the following antimicrobial resistance genes: ESBL producer (bla_CTX–M groups – 1, 2, 8, 9, and 25) (Arlet and Philippon, 1991 (link); Woodford et al., 2005 (link)); and AmpC-type producer (mox, fox, ebc, acc, dha, and cit) (Pérez-Pérez and Hanson, 2002 (link)). The PCR products positive to bla_CTX–M group 1 were characterized for bidirectional Sanger sequencing on ABI-PRISM 3500 XL (Applied Biosystems), following the manufacturer’s recommendations.

Gazal L.E., Medeiros L.P., Dibo M., Nishio E.K., Koga V.L., Gonçalves B.C., Grassotti T.T., de Camargo T.C., Pinheiro J.J., Vespero E.C., de Brito K.C., de Brito B.G., Nakazato G, & Kobayashi R.K. (2021). Detection of ESBL/AmpC-Producing and Fosfomycin-Resistant Escherichia coli From Different Sources in Poultry Production in Southern Brazil. Frontiers in Microbiology, 11, 604544.

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Microsatellite Cross-Amplification of Geophagus brasiliensis

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Cross-amplification tests were conducted from seven loci described for Geophagus brasiliensis (Gbra6, Gbra16, Gbra17, Gbra62, Gbra63, Gbra80, and Gbra96) (Ferreira et al., 2013 (link)). Reagent concentrations and PCR conditions were performed according to Ferreira et al. (2015) (link), using the modifications proposed by Schuelke (2000) (link). PCR thermal conditions were conducted as follows: initial denaturation step at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 40 s. The annealing temperatures of successful cros-amplifications loci were 48°C (Gbra16, Gbra62, Gbra63, Gbra80), 54°C (Gbra06, Gbra17, Gbra70), or 60°C (Gbra96) for 1 min, extension at 72°C for 1 min, followed by a final extension at 72°C for 30 min. PCR products were analyzed on an ABI PRISM 3500-XL automated sequencer (Applied Biosystems) using GeneScan 600 Liz (Applied Biosystems) as a molecular weight marker.

Souza-Shibatta L., Kotelok-Diniz T., Ferreira D.G., Shibatta O.A., Sofia S.H., de Assumpção L., Pini S.F., Makrakis S, & Makrakis M.C. (2018). Genetic Diversity of the Endangered Neotropical Cichlid Fish (Gymnogeophagus setequedas) in Brazil. Frontiers in Genetics, 9, 13.

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16S rRNA Gene Sequencing for Bacterial Identification

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Identification of the isolates was determined by amplification, purification of the PCR product, and sequencing of the 16S rRNA gene, as described previously (Safronova et al., 2015 (link)(Safronova et al., , 2019)) (link). The DNA fragment was sequenced using an ABI PRISM 3500xl genetic analyzer (Applied Biosystems, USA).
The search for homologous sequences and related type strains was performed using the NCBI GenBank database (https://www.ncbi.nlm.nih.gov) and the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was constructed using the MEGA7 program and the neighbour-joining method (Tamura et al., 2011) (link). The evolutionary distances were computed using the maximum composite likelihood model.
The rrs sequences were deposited to the NCBI Gen-Bank database under accession numbers: MT912817-MT912845.

Karlov D., Sazanova A., Кузнецова И.Г., Tikhomirova N., Popova Z.P., Osledkin Y.S., Demidov N., Belimov A.A., & Safronova V.I. (2021). Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic. Biological Communications, 66(1).

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Genetic Analysis of Freshwater Fish

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Seven polymorphic loci were used for A. altiparanae; Asty15, Asty26, Asty23, Asty24, Asty04, Asty16, and Asty12 (Zaganini et al., 2012) and eight were used for G. brasiliensis; Gbra16, Gbra17, Gbra21, Gbra25, Gbra47, Gbra55, Gbra62, and Gbra63 (Ferreira et al., 2013) (link). For A. altiparanae, 86 samples were used for genotyping (33 at UPE, 25 at MPE, and 28 at LPE), along with 82 G. brasiliensis samples (29 at UPE, 23 at MPE, and 30 at LPE).
For genotyping on an automated sequencer, the forward primer of each locus was prepared according to the method described by Schuelke (2000) (link), which allow the labeling of PCR products with fluorescent molecules (FAM, HEX, NED, or PET). PCR was performed as previously described (Ferreira et al., 2013) (link) with specific annealing temperatures as described for each primer (Zaganini et al., 2012; Ferreira et al., 2013) (link). The electrophoresis of PCR products was performed in an automated sequencer, ABI PRISM 3500-XL (Applied Biosystems, Foster City, CA, USA), using the GeneScan 600 Liz (Applied Biosystems) molecular weight marker. Genotypes were determined manually using the GeneMarker 1.85 software (Soft Genetics, State College, PA, USA).

Ferreira D.G., Lima S.C., Frantine-Silva W., Silva J.F., Apolinário-Silva C., Sofia S.H., Carvalho S, & Galindo B.A. (2016). Fine-scale genetic structure patterns in two freshwater fish species, Geophagus brasiliensis (Osteichthyes, Cichlidae) and Astyanax altiparanae (Osteichthyes, Characidae) throughout a Neotropical stream. Genetics and molecular research : GMR, 15(4).

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