Xl 3500 genetic analyzer

Manufactured by Thermo Fisher Scientific

The XL 3500 Genetic Analyzer is a laboratory instrument designed for DNA sequencing analysis. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments. The instrument is capable of processing multiple samples simultaneously, providing high-throughput genetic data for research and diagnostic applications.

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Lab products found in correlation

Qiaamp dna blood mini kit, by Qiagen (2 mentions) Taqman snp genotyping assay, by Thermo Fisher Scientific (2 mentions) Bigdye terminator v3.1 cycle, by Thermo Fisher Scientific (1 mentions) Sequencing buffer, by Thermo Fisher Scientific (1 mentions) Taqpath proamp master mix, by Thermo Fisher Scientific (1 mentions)

3 protocols using xl 3500 genetic analyzer

Sequencing and analysis of TP53, BRCA1, and BRCA2

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For TP53, the different melting curve fragments were sequenced in an automatic sequencer XL 3500 Genetic Analyzer (Applied Biosystems). The reaction consisted of 1 to 2 uL of amplified DNA, 2 uL of Big Dye Terminator v3.1 Cycle, 2 uL of 5X Sequencing Buffer (Applied Biosystems), 1 uL of primer and sufficient water to complete 10 uL. The amplification parameters of the sequencing reaction were: 95°C for 1 minute followed by 25 cycles of 95°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes.
The complete coding sequence and exon-intron boundaries of BRCA1 and BRCA2 were analyzed in two TP53 p.R337H positive females. The sequences of the primers were those described by Leeneer et al. [23 (link)], and Keshavarzi et al. [24 (link)], respectively.

Cury N.M., Ferraz V.E, & Silva WA J.r. (2014). TP53 p.R337H prevalence in a series of Brazilian hereditary breast cancer families. Hereditary Cancer in Clinical Practice, 12(1), 8.

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Genotyping of LIF T/G Polymorphism

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To study the SNP LIF T/G (rs929271), a sample of peripheral venous blood was collected from each participant into an EDTA-containing tube. The DNA was extracted using the QIAamp DNA Blood mini kit (Qiagen). Genotyping was carried out using real-time polymerase chain reaction (PCR) amplification with separate reactions for each sample using a Taqman SNP genotyping assay (Applied Biosystems). The PCR conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 30 s, 60°C for 1 min and 72°C for 1 min.
The samples were assayed in duplicate following the manufacturer's instructions for the chosen SNP. It used a validated TaqMan ® SNP Genotyping Assay (rs929271) in which the context sequence [VIC/FAM] is AACAGTGTGAACCAGC CCCCTGGAA[G/T]CAAGACAGAAAGGCACCCGGCCTCT. Fifteen samples of each genotype were sequenced in an automatic sequencer XL 3500 Genetic Analyzer (Applied Biosystems) to validate the genotyping results. The representative allele discrimination plot (TaqMan ® Genotyper Software) of LIF T/G polymorphism is shown in Figure 1.

Oliveira J.B., Vagnini L.D., Petersen C.G., Renzi A., Oliveira-Pelegrin G.R., Mauri A.L., Ricci J., Massaro F.C., Dieamant F., Cavagna M., Baruffi R.L, & Franco JG J.r. (2016). Association between leukaemia inhibitory factor gene polymorphism and pregnancy outcomes after assisted reproduction techniques. Reproductive biomedicine online, 32(1).

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Genotyping of Population Genetic Variations

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Genomic DNA for the entire studied population was extracted from peripheral blood samples using the QIAamp DNA blood mini kit (Qiagen), following the manufacturer's instructions.
Nucleotide changes were evaluated in duplicate by realtime polymerase chain reaction (PCR) using individual Taq-Man SNP genotyping assays (Thermo Fisher) for each SNP (TP63 rs12486772, VEGFA rs3025010, MMP2 rs2287076, ESR1 rs12199722, ESR2 rs1952585, and LIF rs929271) and TaqPath ProAmp Master Mix, following the manufacturer's instructions, on a StepOne Real-time PCR System. The PCR conditions were as follows: 60 C for 30 seconds (pre-read); 95 C for 5 minutes (initial denature, enzyme activation), 40 cycles of 95 C for 15 seconds (denature), and 60 C for 1 minute (anneal/extend). Genotyping results were validated and confirmed by the automatic sequencer XL 3500 Genetic Analyzer (Applied Biosystems) using 20 samples of each genotype from each polymorphism (normal homozygous, heterozygous, and mutated homozygous) selected randomly. For the minor allele frequency of each polymorphism, all genotypes were sequenced.

Vagnini L.D., Renzi A., Petersen B., Canas M.D.C.T., Petersen C.G., Mauri A.L., Mattila M.C., Ricci J., Dieamant F., Oliveira J.B.A., Baruffi R.L.R, & Franco JG J.r. (2019). Association between estrogen receptor 1 (ESR1) and leukemia inhibitory factor (LIF) polymorphisms can help in the prediction of recurrent implantation failure. Fertility and sterility, 111(3).

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