The mouse Ig L and H chain variable genes of the 24E9 mAb were sequenced to determine the amino acid sequences of the CDRs. cDNA was made from single 24E9 hybridoma cells using a QIAGEN OneStep RT-PCR Kit with degenerate primers designed to target mouse Ig variable regions (25 (link)). cDNA was amplified using Phusion HF polymerase (New England Biolabs), and PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. Sequence data were collected on a 3100-Avant Genetic Analyzer (Applied Biosystems). The nucleotide sequences of 24E9 CDRs can be retrieved from GenBank using accession numbers KJ418726 (H chain) and KJ418727 (L chain) (http://www.ncbi.nlm.nih.gov/genbank ).
3100 avant genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3100-Avant Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing and fragment analysis. It utilizes fluorescent dye-labeled DNA fragments and a laser-induced fluorescence detection system to separate and analyze DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Onestep rt pcr kit, by Qiagen (2 mentions)
Phusion hf polymerase, by New England Biolabs (2 mentions)
Abi prism 3100 avant, by Thermo Fisher Scientific (2 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Abi prism bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (2 mentions)
Lasergene 6, by DNASTAR (1 mentions)
Titan one tube rt pcr system, by Roche (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiamp viral rna mini kit, by Qiagen (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Gfx pcr dna gel band purification kit, by GE Healthcare (1 mentions)
Abi 3500dx sequencer, by Thermo Fisher Scientific (1 mentions)
Powerplex 16 kit, by Promega (1 mentions)
Nucleon bacc3 genomic dna extraction kit, by GE Healthcare (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Simpleprep reagent for dna, by Takara Bio (1 mentions)
Nucleospin plant kit, by Macherey-Nagel (1 mentions)
16 protocols using 3100 avant genetic analyzer
1
Sequencing Mouse Antibody Variable Genes
2
Viral Genome Sequencing from Chicken Embryos
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Viral RNA was isolated from the allantoic fluid of infected chicken embryos with a commercial QIAamp Viral RNA mini kit (QIAGEN, Hilden, Germany). Full-length viral genome segments were obtained by reverse transcription and PCR with specific terminal primers, MMLV, and Taq-polymerase (Alpha-Ferment Ltd., Moscow, Russia). The amplified fragments were separated by electrophoresis in 1–1.3% agarose gel and extracted from the gel with the Diatom DNA elution kit (Isogene Laboratory Ltd., Moscow, Russia, # D1031). Sequencing reactions were performed with terminal or internal primers with the BrightDye™ Terminator Cycle Sequencing Kit v3.1 (Nimagen, the Netherlands) followed by analysis on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems 3100-Avant Genetic Analyzer, Foster City, CA, USA). The Lasergene 6 software package (DNASTAR Inc., Madison, WI, USA) was used for assembly and analysis of nucleotide sequences.
3
Viral RNA Extraction and Sequencing
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Viral RNA was extracted from 140 µl plasma using QIAmp Viral RNA Mini Kit (Qiagen). RT-PCR was performed with Titan One Tube RT-PCR System (Roche). Nested PCR was done using an in-house method described elsewhere [15] (link), [20] (link). Thermal cycling conditions for PCR and primers sequence and position were previously described [15] (link), [20] (link). DNA sequences were obtained with Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and an automated sequencer (3100-Avant Genetic Analyzer, Applied Biosystems).
4
Viral Genome Sequencing from Allantoic Fluid
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Viral RNA was isolated from the allantoic fluid of infected chicken embryos with a commercial QIAamp Viral RNA mini kit (Qiagen, # 52904). Full-length viral genome segments were obtained by reverse transcription and PCR with specific terminal primers, MMLV, and Taq-polymerase (Alpha-Ferment Ltd., Moscow, Russia). The amplified fragments were separated by electrophoresis in 1–1.3% agarose gel and subsequently extracted from the gel with the Diatom DNA Elution kit (Isogene Laboratory Ltd., Moscow, Russia, # D1031). Sequencing reactions were performed with terminal or internal primers with the BrightDye™ Terminator Cycle Sequencing Kit v3.1 (Nijmegen, The Netherlands), followed by analysis on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems 3100-Avant Genetic Analyzer, Foster City, CA, USA). The Lasergene software package (DNASTAR Inc., Madison, WI, USA) was used for the assembly and analysis of nucleotide sequences.
5
Sequencing Mouse Immunoglobulin Variable Genes
6
Amplification and Sequencing of HIV-1 env
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Chromosomal DNA was extracted from infected peripheral blood mononuclear cells of each subject using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer recommendations. Nested PCR was done to obtain a 534 bp fragment from the C2V3 env region (HXB2 positions 6858–7392). Thermal cycling conditions were as previously described (Bartolo et al. 2009 (link)). PCR products were cloned into the pCR4-TOPO vector (Invitrogen), using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s instructions. DNA sequencing was performed using the BigDye Terminator V3.1 Cycle sequencing Kit (Applied Biosystems, Foster City, CA) and an automated sequencer (3100-Avant Genetic Analyzer; Applied Biosystems). We derived 31, 20, and 19 sequences from MP1, MP2, and MP3, respectively.
7
DNA Sequencing Purification Protocol
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Amplification products were purified by electrophoresis (Sambrook, Fritsch & Maniatis, 1989 ), one-band DNA fragments were extracted from the gel and purified using the GFX™ PCR DNA, Gel Band Purification Kit (GE HealthCare, Little Chalfont, UK), or Evrogene Cleanup Mini (Russia), and then used as a template in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) following the standard protocol provided for 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fragment sequences were determined at the Genom Center (Engelhardt Institute of Molecular Biology RAS, Moscow, Russia).
8
Bidirectional DNA Sequencing Protocol
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A bidirectional direct sequencing of polymerase chain reaction (PCR) products by direct sequencing of exons 1 to 52, including exon/intron boundaries, 5′ and 3′ untranslated region, was performed with an ABI 3500Dx Sequencer (Applied Biosystems), as previously detailed.28 The paternity test was performed with the Power‐Plex‐16 kit (Promega Corp.) following the manufacturer’s instructions. Analysis of the PCR products (3 μl) was carried out with the 3100‐Avant Genetic Analyzer (Applied Biosystems). The quality control followed the proficiency testing of International Society for Forensic Genetics Working Group guidelines.29
9
Tumor Genomic DNA Extraction and Sequencing
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The DNA extraction procedures and DNA sequencing methods used in this study have been previously described.11 , 19 Genomic DNA was isolated from primary tumor samples by overnight digestion with SDS and proteinase K at 37°C, followed by standard phenol‐chloroform extraction and ethanol precipitation. Exons 5–8 p53 gene mutations and exons 19 and 21 EGFR gene mutations were determined by direct sequencing. The primers used for detection of both gene mutations were according our previous reports.11,19 All polymerase chain reaction products were incubated with exonuclease 1 and shrimp alkaline phosphatase and then sequenced directly using an automated sequencing system (3100 Avant Genetic Analyzer; Applied Biosystems, Hitachi, Japan).
10
Genotype Assessment Protocol
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Informed consent for genotype assessment was obtained from patients (or parents) for being included in the study. Genotyping analyses were conducted in a single laboratory. Genomic DNA was extracted from blood samples using the Nucleon BACC3 Genomic DNA Extraction Kit (GE Healthcare). PCR was carried out with 13 sets of oligonucleotides amplifying all 13 exons of the gene and the exon-intron junctions. Amplicons were separated by dHPLC as previously reported [23 (link)]. Fragments showing an abnormal dHPLC pattern were purified (Qiaquick PCR purification kit, Qiagen), quantified and sequenced using the ABI PRISM Big Dye terminator V1.1 sequencing kit and the 3100-Avant Genetic Analyzer (Applied Biosystems). When no mutation was identified with dHPLC, all exons were sequenced.
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