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Applied biosystems geneamp pcr system 9700

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Applied Biosystems GeneAmp PCR System 9700 is a thermal cycler designed for performing polymerase chain reaction (PCR) experiments. It has the capability to precisely control temperature and timing of the thermal cycling process, which is essential for DNA amplification and analysis.

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13 protocols using applied biosystems geneamp pcr system 9700

1

Isolation and qPCR Analysis of Macrophage RNA

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Total RNA was isolated from myometrial and decidual macrophages using the RNeasy mini kit (Qiagen), following the manufacturer's instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies, Wilmington, DE, USA) using the Agilent RNA 6000 Pico Kit (Agilent). cDNA was synthesized by using iScript Reverse Transcription Supermix for RT-qPCR kits (Bio-Rad Laboratories, Hercules, CA, USA) on the Applied Biosystems GeneAmp PCR System 9700 (Life Technologies), following the manufacturer's instructions. cDNA was amplified using the SsoAdvanced PreAmp Supermix (Catalog #1725160; Bio-Rad Laboratories) on the Applied Biosystems GeneAmp PCR System 9700. mRNA expression of Nfκb1, Tnf, Il10, and housekeeping genes (Actb, Gapdh, and Tbp) was determined by qPCR using the LuminoCtÒ SYBR Green qPCR ReadyMix (Sigma Aldrich) on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories), according to the manufacturer's instructions. Primers are described in Supplementary Table II.
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2

Transcriptomic Analysis of Decidual and Myometrial Tissues

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Dams were injected with αCD3ε, LPS, or RU486 (or their respective controls) (n = 7 – 9 each for αCD3ε, 9 – 13 each for LPS, and 11 – 16 each for RU486). Mice were euthanized 12–16 h post-injection and decidual and myometrial tissues from the implantation sites were collected and placed in RNAlater Stabilization Solution (Life Technologies). Total RNA was isolated from decidual and myometrial tissues using the RNeasy mini kit (Qiagen), following the manufacturer’s instructions. RNA concentrations and purity were assessed with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was evaluated with the 2100 Bioanalyzer system (Agilent Technologies) using the Agilent RNA 6000 Nano Kit (Agilent Technologies). Complementary (c)DNA was synthesized by using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies) on the Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies), following the manufacturer’s instructions. Complementary DNA was amplified using the TaqMan® PreAmp Master Mix (2X) (Applied Biosystems) on the Applied Biosystems 7500 Fast Real-time PCR System. Messenger RNA expression was determined by quantitative real-time PCR (qRT-PCR) using a BioMark high-throughput qRT-PCR System (Fluidigm) and TaqMan® gene expression assays (Thermo Fisher) (Supplementary Table I).
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3

Amplification and Sequencing of MC1R and KIT Genes

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The primers were designed to amplify exon 1, which was the target for the MC1R gene, and exons 2 and 3, which were the targets for the KIT gene (Table-1) [5 , 13 ]. Polymerase chain reaction (PCR) amplification was conducted using GoTaq® Green Master Mix (PROMEGA Corporation, USA) with a total volume of 25 μL containing 25 pm/mL forward and reverse primer, 10−100 ng/μL DNA template, and nuclease-free water. The PCR reaction was performed on an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific Inc. USA) with initial denaturation at 95°C for 5 min (1 cycle), denaturation at 95°C for 10 s (35 cycles), annealing for each target fragment (exon 1 for MC1R; exons 2 and 3 for KIT), and elongation at 72°C for 5 min (1 cycle). Polymerase chain reaction products from each fragment were visualized using electrophoresis in 1.5% agarose gel, stained with Floro Safe DNA (Axil Scientific Pte Ltd., Singapore), and documented using an ultraviolet transilluminator (AlphaImaginer, Alpha Inotech, Santa Clara, USA). The PCR products of MC1R and KIT were sent to 1st BASE, Selangor, Malaysia, for sequencing.
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4

Investigating QS Regulation in P. aeruginosa

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Wild-type PA01 and isogenic ∆lasIrhlI mutant cultured to different growth phases (as described above) were inoculated into mouse lungs or under in vitro conditions (BALF, LB, 0.9% NaCl) for a predetermined amount of time. Total RNA was extracted from bacteria pellets or lung homogenates by using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). cDNA was prepared from the same amount of total RNA as the templates by TaqMan® Real-Time PCR Assays (Thermo Fisher Scientific), with the probes and primers for the lasI, lasR, rhlI, rhlR, lasB and rhlA genes designed by the Applied Biosystems. qPCR wasperformed on an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). The P. aeruginosa housekeeping gene rpsL was used as endogenous control. All the experiments were independently performed three times in triplicate.
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5

Genetic Profiling of Tumor Specimens

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Tumour DNA was extracted from tumour specimens with NucleoSpin® Tissue (MACHEREY-NAGEL, Düren, Germany). Regions of interest for driver genes [23 (link), 27 (link)–30 (link)] were amplified by PCR with gene-specific primers (Supplementary Table 1) and TaKaRa Ex Taq® (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp, KRAS, HRAS, and NRAS) using Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were purified by ExoSAP-IT (Affymetrix, Santa Clara, CA), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp, and exons 2 and 3 of KRAS, HRAS, and NRAS) and BigDye® Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).
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6

Genetic Analysis of MADD Patients

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Two male MADD patients, one relative from the affected pedigree and one normal control from an unrelated pedigree were included. This study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects. The protocol was approved by the Ministry of Science and Technology of Taiwan and the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB-N201506002). Whole blood (15 mL) from the study participants was drawn and collected in EDTA-containing tubes. Genomic DNA was isolated from the blood cells using a DNA purification kit (QIAamp DNA Mini kit, Qiagen, Valencia, CA, USA). Primer pairs covering 13 coding exons and the flanking intron splice sites were prepared and used to amplify DNA segments by polymerase chain reaction (PCR) using a DNA thermal cycler (Applied Biosystems GeneAmp PCR system 9700, Thermo Fisher Scientific, Foster City, CA, USA). The PCR products were purified and mixed with a dye terminator cycle sequencing kit (Applied Biosystems) and sequenced using an auto sequencer (Applied Biosystems 3730XL DNA Analyzer, Thermo Fisher Scientific). The putative mutations were tested for segregation in the family by direct sequencing.
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7

Synthesizing cDNA from RNA Samples

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cDNA was synthesized using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific) according to a modified version of the manufacturer’s instructions. First, 2 μl of SuperScript™ IV VILO™ Master Mix was added to 8 μl of extracted RNA (100 ng total RNA) in reaction tubes. Reverse transcription was conducted by heating the reaction mixture to 25 °C for 10 min, 50 °C for 10 min, and 85 °C for 5 min, before cooling to 4 °C with the Applied Biosystems GeneAmp PCR system 9700 (Thermo Fisher Scientific). As the majority of samples were small, the minimum amount of cDNA that could be detected by PCR probes was determined in advance, as was the dilution concentration. Synthesized cDNA was diluted 10-fold with 10 mM Tris-HCl (pH 8.0) and stored at −70 °C.
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8

SNP Genotyping for Sample Identification

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DNA samples were normalized to 5–10 ng/μl, and identity test was performed using iPLEX® Pro Sample ID Panel kit (Cat. No. 25094, Agena Bioscience, San Diego, CA). Multiplex-PCR targeting 44 identity SNPs was carried out in a 5-μl volume on an Applied Biosystems GeneAmp® PCR System 9700 (Thermo Fisher Scientific, Waltham, MA) with the cycling program recommended by the manufacturer’s manual. After PCR, amplicons were treated with 5 U shrimp alkaline phosphatase (SAP; Agena Bioscience, San Diego, CA USA) and followed by adding 2 μl single-base extension (SBE) cocktail (Agena Bioscience, San Diego, CA) to continue the SBE reaction on a GeneAmp® PCR System 9700 with the cycling program recommended by the manufacturer. SBE products were cleaned up using an ion-exchange resin and then spotted on SpectroCHIP arrays (Agena Bioscience, San Diego, CA USA). Raw data were acquired on a MassARRAY® 4 system (Agena Bioscience, San Diego, CA USA) by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. Genotypes for 44 SNPs were generated by MassARRAY Typer software v4.0 (Agena Bioscience, San Diego, CA).
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9

Sequencing of Tumor Driver Genes

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Tumour DNA was extracted from tumour specimens using NucleoSpin Tissue (MACHEREY–NAGEL, Düren, Germany). Regions of interest for driver genes3 (link),16 (link)–18 (link) were amplified by PCR with gene-specific primers (Supplementary Table S1) and TaKaRa Ex Taq (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp) using an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were processed by ExoSAP-IT (Thermo Fisher Scientific), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp) and a BigDye Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).
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10

DNA Profiling of Buccal Swab Samples

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Referent samples (12 buccal swabs) from potential living relatives were collected, recorded, and preliminarily labeled by local DNA experts. Living relatives were directly contacted by a local person (Mr Jure Lauc), who was present during the collection of each sample. Collection was performed with an Internal GENOS collection kit, and dried and labeled samples were transported to the laboratory. Upon arrival, the samples were relabeled and the relevant information entered into the Chain of Custody forms. The samples were stored at -80°C until DNA extraction was performed using the QIAmp DNA Mini Kit (14 ).
PowerPlex® ESI kit (Promega Corp.) was used for further analysis. Similar amounts of DNA were used in all PCR reactions. Amplification was carried out as described previously (12 ). The total reaction volume was 10 μL. Additionally, PowerPlex®Y23 kit (Promega Corp.) was used to simultaneously amplify 23 Y-STR loci according to manufacturer’s recommendations (13 ). PCR amplification was carried out by the Applied Biosystems® GeneAmp® PCR System 9700 (Life Technologies) according to manufacturer’s recommendations. Electrophoresis of the amplification products was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The raw data were compiled and analyzed using 310 Data Collection Software and GeneMapperTM 3.2.
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