3730 dna genetic analyzer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 3730 DNA Genetic Analyzer is a capillary electrophoresis-based instrument designed for high-throughput DNA sequencing and fragment analysis applications. It utilizes laser-induced fluorescence detection to analyze DNA samples. The instrument can process multiple samples simultaneously and generate accurate DNA sequence data or fragment size information.

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Lab products found in correlation

Rox 500, by Thermo Fisher Scientific (3 mentions) Stop buffer, by Promega (2 mentions) Dye primer based sequencing kit, by Thermo Fisher Scientific (2 mentions) Gentra puregene cell kit, by Qiagen (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)

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3730 DNA Analyzer (393 citations) 3730 Genetic Analyzer (85 citations)

4 protocols using 3730 dna genetic analyzer

Genetic Analysis of TAZ Mutations in BTHS

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All seven patients had TAZ mutation analysis performed as part of their investigations for BTHS or for family studies. Consent for genetic testing was obtained at the time of sample collection.
Genomic DNA was received from an external laboratory or isolated from peripheral blood leukocytes using a Gentra Puregene cell kit (QIAGEN Ltd). Coding regions of the TAZ gene (11 exons including intron/exon boundaries extending to the branch sites) were amplified in ten fragments using a MegaMix (MicroZone) and GC-RICH (Roche) PCR system. Primers were designed to GenBank Reference Sequence NM_000116.3 using Primer3 software (Koressaar and Remm 2007 (link); Untergasser et al 2012 ). M13 tagged bi-directional sequencing was undertaken using a BigDye Terminator v3.1 Cycle Sequencing Kit and 3730 DNA Genetic Analyzer (Applied Biosystems) with Mutation Surveyor DNA Variant Analysis Software v3.97 (Softgenetics). Alamut software v2.3.1 (Interactive Biosoftware, Rouen, France) was used to predict the effect of genetic variation. The software integrates PolyPhen-2, Align GVGD and SIFT and five splice site prediction programs: SpliceSiteFinder, MaxEntScan, Human Splice Finder, NNSPLICE and GeneSplicer.

Bowron A., Honeychurch J., Williams M., Tsai-Goodman B., Clayton N., Jones L., Shortland G.J., Qureshi S.A., Heales S.J, & Steward C.G. (2014). Barth syndrome without tetralinoleoyl cardiolipin deficiency: a possible ameliorated phenotype. Journal of Inherited Metabolic Disease, 38(2), 279-286.

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Fluorescence-based DNase I Footprinting

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DNase I footprinting assays were performed based on a fluorescence labeling procedure56 (link). Briefly, the promoters DNA of exosporium genes were PCR-amplified using the fluorescently labeled primers and purified from an agarose gel. The labeled DNA probe (400 ng) was incubated for 30 min at 25 °C with the different amounts of GerE in a total volume of 40 μl binding buffer (described above for EMSA). DNase I digestion was then performed for 1 min at 25 °C and stopped with stop buffer (Promega). After phenol-chloroform extraction and ethanol precipitation, the samples were loaded on an Applied Biosystems 3730 DNA genetic analyzer with an internal-lane size standard (ROX-500, Applied Biosystems). A dye primer-based sequencing kit (Thermo) was used to precisely determine the sequences after their alignment wtih capillary electrophoresis results. Electropherograms were analyzed with GeneMarker v1.8 (Applied Biosystems).

Peng Q., Kao G., Qu N., Zhang J., Li J, & Song F. (2016). The Regulation of Exosporium-Related Genes in Bacillus thuringiensis. Scientific Reports, 6, 19005.

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Fluorescent DNase I Footprinting Assay

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DNase I footprinting assays were performed based on a fluorescent labeling procedure [23 (link)]. Briefly, gabT promoter DNA was PCR-amplified using the fluorescently-labeled primers PgabT-F and PgabT-R (Table 2) and purified from an agarose gel. The labeled PgabT DNA probe (120 ng) was incubated for 20 min at 25°C with the indicated concentrations of purified GabR and BSA in a total volume of 50 μl of binding buffer (described above for EMSA). DNase I digestion was then carried out for 1 min at 25°C and stopped with stop buffer (Promega). After phenol-chloroform extraction and ethanol precipitation, the samples were loaded on an Applied Biosystems 3730 DNA genetic analyzer together with an internal-lane size standard (ROX-500, Applied Biosystems). A dye primer-based sequencing kit (Thermo) was used to precisely determine the sequences after their alignment wtih the capillary electrophoresis results from the reactions. Electropherograms were analyzed with GeneMarker v1.8 (Applied Biosystems).

Peng Q., Yang M., Wang W., Han L., Wang G., Wang P., Zhang J, & Song F. (2014). Activation of gab cluster transcription in Bacillus thuringiensis by γ-aminobutyric acid or succinic semialdehyde is mediated by the Sigma 54-dependent transcriptional activator GabR. BMC Microbiology, 14, 2317.

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DNase I Footprinting of SAV577 Protein

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This assay was performed using an FAM fluorescence labeling capillary electrophoresis method [28] (link). Two fluorescence-labeled DNA fragments amplified with primer pairs FAM-GJ78/GJ77 and FAM-GJ228/GJ227 (Table S1) were used to characterize the binding sites of SAV577 protein in the SAV575-SAV576 intergenic region. The resulting 547-bp and 478-bp DNA fragments covered the entire intergenic region. The labeled DNA fragments (400 ng) and corresponding concentrations of His₆-tagged SAV577 protein were added to a reaction mixture (final volume 50 µl) and incubated for 30 min at 25°C in binding buffer [20 mM HEPES (pH 7.6), 10 mM (NH₄)₂SO₄, 1 mM DTT, 0.2% Tween-20, 30 mM KCl]. DNase I (0.016 units) digestion was performed for 40 s at 37°C and stopped by addition of EDTA at a final concentration of 60 mM. The reaction mixture was heated to 80°C for 10 min to totally inactivate DNase I. Samples were subjected to phenol-chloroform extraction, ethanol precipitation, and capillary electrophoresis by loading into an 3730 DNA Genetic Analyzer with the internal-lane size standard ROX-500 (Applied Biosystems, USA). Electrophoregrams were analyzed using the GeneMarker program, v1.8 (Applied Biosystems).

Guo J., Zhang X., Chen Z., Wen Y, & Li J. (2014). Two Adjacent and Similar TetR Family Transcriptional Regulator Genes, SAV577 and SAV576, Co-Regulate Avermectin Production in Streptomyces avermitilis. PLoS ONE, 9(6), e99224.

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