Screening of XLID families linked to the Xq12 region of the X chromosome was conducted using M13 tagged primers for the exons of ZC4H2. The PCR products were amplified and then purified with ExoSap from USB. Using M13 primers and the ABI 3730 sequencer the PCR products were sequenced with BIGDye Terminator v3.1 Cycle sequencing Kit from Life Technologies. The sequence data were analyzed using SEQMAN from DNASTAR. The R213W alteration was found in an affected male and then tested and confirmed in the entire family.
3730 sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The 3730 DNA Analyzer is a capillary electrophoresis-based genetic analysis system designed for high-throughput DNA sequencing. The system provides accurate and reliable data for a wide range of applications, including genome sequencing, fragment analysis, and genetic profiling.
Automatically generated - may contain errors
Lab products found in correlation
High pure viral rna kit, by Roche (3 mentions)
One step rna pcr kit, by Takara Bio (3 mentions)
Bigdye terminator v1, by Thermo Fisher Scientific (2 mentions)
Faststart high fidelity pcr system, by Roche (2 mentions)
Sequencer v 4, by Gene Codes (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Seqman, by DNASTAR (1 mentions)
Taq pcr core kit, by Qiagen (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Genemapper software version 4, by Thermo Fisher Scientific (1 mentions)
Puregene protocol, by Qiagen (1 mentions)
Chromas software, by Technelysium (1 mentions)
Dnaman 8, by Lynnon Biosoft (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Bigdye terminator cycle sequencing ready reaction kit version 3, by Thermo Fisher Scientific (1 mentions)
Agencourt ampure xp purification system, by Beckman Coulter (1 mentions)
Kod dna polymerase, by Toyobo (1 mentions)
Rtaq kit, by Takara Bio (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
One step rt pcr kit, by Takara Bio (1 mentions)
26 protocols using 3730 sequencer
1
Genetic Screening for XLID Variants
2
MSI Determination in TCGA and HMUCH Cohorts
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MSI status for the TCGA cohort was determined using the MSI sensor (version 0.5). In brief, for MSI sensor scores < 3.5, samples were considered to be MSS; otherwise, they were considered MSI (27 (link)). Published studies using the TCGA cohort provided FS-mutation and TMB data (28 (link)–30 (link)), and MSI status for the HMUCH cohort was determined using a 3730 sequencer (Life Technologies, Carlsbad, CA, USA). For this purpose, whole blood samples or prepared FFPE tissue were diluted to 2 ng/μL or 20 ng/μL, respectively, followed by the addition of 2.8 μL of ddH2O, 4 μL of 2.5× Buffer A, 2 μL of 5× MSI Primer Mix, and 0.2 μL of Taq DNA Polymerase I. PCR amplification was carried out as follows: pre-denaturation at 95°C for 5 min; followed by 30 cycles at 94°C for 30 s, 60°C for 1 min, and 70°C for 1 min; and then a final extension at 60°C for 30 min. Finally, the temperature was reduced to 15°C, and the samples were centrifuged at 3,000 × g for 1 min. NR-21 and BAT-26 were labeled with blue fluorescent dye, BAT-25 with green dye, and NR-24 and MONO-27 with yellow dye. Finally, tumors were classified as MSI-H if two or more markers showed instability; otherwise they were classified as MSS.
3
MSI Detection Protocol for Whole Blood and FFPE
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MSI status was determined using a 3730 sequencer (Life Technologies, Carlsbad, CA). For this purpose, whole blood samples (1 μL) or prepared FFPE tissue were diluted to 2 ng/μL or 20 ng/μL, respectively, followed by addition of 2.8 μL ddH 2 O, 4 μL 2.5× Buffer A, 2 μL 5× MSI Primer Mix, and 0.2 μL Taq DNA Polymerase I. PCR amplification was carried out as follows: Pre-denaturation at 95°C for 5 min; followed by 30 cycles at 94°C for 30 s, 60°C for 1 min, 70°C for 1 min; and then final extension at 60°C for 30 min. Finally, the temperature was reduced to 15°C, and samples were centrifuged at 3000 rpm for 1 min. NR-21 and BAT-26 were labeled with a blue fluorescent dye, NR-27 and BAT-25 with a green dye, and NR-24 and MONO-27 with a yellow dye. Tumors were termed MSI-H if two or more markers showed instability, MSI-L if only one marker was unstable, and MSS in case of no mutation.
4
HIV Pol Gene Sequencing from Plasma
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Total viral RNA was extracted from plasma isolated from blood samples using the Roche High Pure Viral RNA kit (Roche, REF: 11858882001). A nested reverse transcriptase polymerase chain reaction (RT-PCR) was applied to amplify a pol fragment of the 1062 bp length region (from 2253 to 3314 according to HXB2 calibrator) spanning the protease gene and partial reverse transcriptase gene using the One Step RNA PCR Kit (Takara, RR055A) and the ExTaq Kit (Takara, RR902A) with sets of primers and thermal cycling conditions as described previously [20 (link)]. The PCR products were purified and subjected to direct DNA sequencing on an Applied Biosystems 3730 Sequencer.
5
COI mtDNA Gene Amplification Protocol
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A species-specific set of primers developed by Katsares et al.52 (link) were used for amplification of a fragment of the COI mtDNA gene. PCRs were performed using Taq PCR Core Kit (Qiagen, Hilden, Germany) in a volume of 23 µL containing 2.5 µL of 10× PCR Buffer, 2 µL of MgCl2 (25 mM), 2.5 µL of dNTP mix (2 mM), 1 µL of each primer (10 µM), 0.1 µL of TAQ DNA polymerase (5 U/µL), 11.9 µL of RNA free water and 2 µL of DNA template (5 ng/µL). Thermal cycling of PCR consisted of an initial denaturing step of 3 min at 94 °C, followed by 40 cycles of amplification (1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C) and a final extension step of 3 min at 72 °C. PCR products were then sent to GenoScreen (Lille, France) to be sequenced on an Applied Biosystems 3730 Sequencer.
6
Targeted IDH Mutation Sequencing
7
Microsatellite Genotyping of Gray Reef Sharks
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Total DNA was extracted from fin clips using the PureGene protocol (Qiagen, Hilden, Germany). A total of 15 microsatellite markers developed for gray reef sharks were amplified using the primers and cycling parameters from Momigliano et al.57 (link). PCR products were sent for sequencing to an external private company (GenoScreen, Lille, France) to be run on an Applied Biosystems 3730 sequencer and were genotyped employing GeneMapper software version 4.0 (Applied Biosystems, Foster City, CA, USA).
8
Investigating Species Relationships via COI Barcoding
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To determine if our increased sampling effort was enough to resolve the relationships of these two species, we amplified all samples for the mitochondrial COI barcoding region. Primers and PCR reactions protocols were identical those described in Ludt et al. (2015 ) and can be found in the appendix. All samples were purified and sequenced in both forward and reverse directions using the Genomic Sequencing and Analysis Facility at the University of Texas at Austin. Sequencing was performed on an Applied Biosystems 3730 sequencer. All sequences were edited and aligned using Geneious 6.0.5 (Biomatters), and all alignments were checked manually. Haplotype networks were created using the TCS networks option in PopART (Clement, Posada, & Crandall, 2000 ). Summary statistics (haplotype and nucleotide diversities, ΦST), and Fu's F statistic (Fu, 1997 ) were calculated using Arlequin 3.5 (Excoffier, Laval, & Schneider, 2005 ). An AMOVA was conducted to test for population structuring between the two species, as well as between sampling localities, using 50,000 permutations in Arlequin. These summary statistics were calculated for both species and for all sampling locations.
9
PCR Product Sequencing Workflow
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Sequencing of PCR products from genomic and cDNA from different mouse strains was performed in the DNA Core Facility of the Kimmel Cancer Center at Thomas Jefferson University using an Applied Biosystems 3730 Sequencer (Applied Biosystems, Foster city, CA). Results were visualized with Chromas software (Technelysium, South Brisbane, Australia).
10
Phylogenetic Analysis of Candidate Genes
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Reference sequences of the candidate genes located in the mapping region were retrieved from the S. italica genome project V2.2. Genes in the mapped region were PCR amplified and the PCR products were sequenced using an Applied Biosystems 3730 sequencer (Applied Biosystems, Foster City, CA, USA) and analysed by DNAMAN8 software (Lynnon Biosoft, Quebec, Canada). Alignments of full-length candidate protein sequences used for phylogenetic analysis were produced by CLUSTALW (Thompson et al., 2002 (link)). The phylogenetic tree was constructed using MEGA5.0 software (Tamura et al., 2011 (link)) and the neighbor-joining method, with 1000 bootstrap value trials. The alignment file is included in Supplementary Table S2 .
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