High sensitivity dna assay kit

Manufactured by Agilent Technologies

Sourced in United States

The High Sensitivity DNA Assay kit is a laboratory equipment product designed for the quantification of DNA samples. It provides a sensitive and accurate method for measuring low concentrations of DNA.

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High Sensitivity DNA Assay (44 citations) Agilent 2100 High Sensitivity DNA Assay Kit (9 citations) Agilent high sensitivity DNA assay kit (5 citations) Bioanalyzer High Sensitivity DNA Assay kit (4 citations) 2100 Bioanalyzer High Sensitivity DNA assay Kit (3 citations) Qubit dsDNA HS(high-sensitivity) Assay Kit on Agilent High Sensitivity DNA Bioanalyzer (2 citations)

15 protocols using high sensitivity dna assay kit

Whole Exome Sequencing from DNA

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QIAamp DNA Blood Mini Kit (Qiagen) was used for DNA extraction from PBMC and fresh tissue. The preparation was performed following the manufacturer's protocol. Starting from 200 ng of DNA from fresh tissue/PBMCs, NGS libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) according to the manufacturer's protocol. Subsequent whole exome target enrichment was performed following IDT xGen protocol (xGen Hybridization and Wash Kit, xGen Universal Blockers-NXT Mix, xGen Exome Research Panel v2; IDT, Inc.). The quality of libraries was checked with the High-Sensitivity DNA assay kit (Agilent Technologies). Library preparation, enrichment of whole exome regions, and sequencing of patient samples were performed by Cogentech Società Benefit srl.
NGS libraries for cell line samples were prepared starting with 150 ng of DNA and processed with Illumina DNA Prep with Enrichment and Exome Panel 45 Mb (Illumina) according to the manufacturer's protocol. After the fragmentation of gDNA with transposon enzyme and subsequent PCR to introduce unique sample indexes, DNA fragment size distribution was assessed using the 2100 Bioanalyzer with a High-Sensitivity DNA assay kit (Agilent Technologies). Equal amounts of DNA libraries were pooled and subjected to targeted panel hybridization capture.
Final libraries were sequenced on NextSeq sequencer 500 or 550 DX (Illumina).

Crisafulli G., Sartore-Bianchi A., Lazzari L., Pietrantonio F., Amatu A., Macagno M., Barault L., Cassingena A., Bartolini A., Luraghi P., Mauri G., Battuello P., Personeni N., Zampino M.G., Pessei V., Vitiello P.P., Tosi F., Idotta L., Morano F., Valtorta E., Bonoldi E., Germano G., Di Nicolantonio F., Marsoni S., Siena S, & Bardelli A. (2022). Temozolomide Treatment Alters Mismatch Repair and Boosts Mutational Burden in Tumor and Blood of Colorectal Cancer Patients. Cancer Discovery, 12(7), 1656-1675.

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Ion Amplicon Library Sequencing

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Amplicon libraries were prepared using an Ion AmpliSeq TM Library Kit Plus (Cat. No. A35907;
Thermo Fisher Scientific, MA, USA). Library quality was assessed using a 2100 Bioanalyzer with a DNA high sensitivity assay kit (Agilent CA, USA). Libraries were quantified using the Ion Library TaqMan TM Quantitation kit (Cat. No. 4468802; Thermo Fisher Scientific, MA, USA). Sequencing was performed on an Ion S5 Plus system using 530 chip and 400bp chemistry.

Soni T., Pandit R., Blake D.P., Joshi C.G., & Joshi M. (2021). Comparative analysis of two NGS platforms and different databases for analysis of AMR genes.

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Ion AmpliSeq Library Preparation

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Amplicon libraries were prepared using an Ion AmpliSeq TM Library Kit Plus (Cat. No. A35907; Thermo Fisher Scientific, MA, USA). Library quality was assessed using a 2100 Bioanalyzer with a DNA high sensitivity assay kit (Agilent CA, USA). Libraries were quantified using the Ion Library TaqMan TM Quantitation kit (Cat. No. 4468802; Thermo Fisher Scientific, MA, USA). Sequencing was performed on an Ion S5 Plus system using 530 chip and 400bp chemistry.

Pietschmann J., Guinat C., Beer M., Pronin V., Tauscher K., Petrov A., Keil G, & Blome S. (2015). Course and transmission characteristics of oral low-dose infection of domestic pigs and European wild boar with a Caucasian African swine fever virus isolate. Archives of virology, 160(7).

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Comprehensive RNA Extraction and Sequencing

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Total RNA was extracted from the 50 human retina samples using the miRNeasy Kit (QIAGEN) according to the manufacturer's instructions. RNA was quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies) and the integrity was evaluated using an RNA 6000 Nano chip on a Bioanalyzer (Agilent Technologies). The RNA of the 50 samples had an average RNA integrity number (RIN) of 8.7 (ranging from 7.2 to 9.7). Libraries were prepared according to manufacturer's instructions (TruSeq RNA Sample Preparation kit) with an initial amount of 4 μg of total RNA. Quality control of library templates was performed using a High Sensitivity DNA Assay kit (Agilent Technologies) on a Bioanalyzer (Agilent Technologies). Qubit quantification platform was used to normalize samples for the library preparation (Qubit 2.0 Fluorometer, Life Technologies). Libraries were sequenced via a paired-end chemistry on an Illumina HiSeq1000 platform with an average yield of ∼6 Mb.

Pinelli M., Carissimo A., Cutillo L., Lai C.H., Mutarelli M., Moretti M.N., Singh M.V., Karali M., Carrella D., Pizzo M., Russo F., Ferrari S., Ponzin D., Angelini C., Banfi S, & di Bernardo D. (2016). An atlas of gene expression and gene co-regulation in the human retina. Nucleic Acids Research, 44(12), 5773-5784.

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Illumina TruSeq™ Stranded mRNA Sequencing

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rRNA-depleted samples were used as input for cDNA library preparation using the Illumina TruSeq™ stranded mRNA kit with a slightly modified library preparation protocol. cDNA libraries were quantified using the Qubit® High Sensitivity DNA assay kit and fragment sizes were determined using the High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. The libraries were normalised to 4 nM and pooled together. Paired-end sequencing was performed using two Miseq reagent kits v3 (150-cycle) on an Illumina MiSeq™ sequencer.

Simpson D.H., Hapeshi A., Rogers N.J., Brabec V., Clarkson G.J., Fox D.J., Hrabina O., Kay G.L., King A.K., Malina J., Millard A.D., Moat J., Roper D.I., Song H., Waterfield N.R, & Scott P. (2019). Metallohelices that kill Gram-negative pathogens using intracellular antimicrobial peptide pathways †Electronic supplementary information (ESI) available: Data created during this study are openly available from the University of Warwick Research Archive Portal (WRAP) (http://wrap.warwick.ac.uk). CCDC 1904782–1904784. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c9sc03532j. Chemical Science, 10(42), 9708-9720.

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Nextera XT DNA Library Preparation for MiSeq

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Preparation of MiSeq library was performed using Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) as previously described with minor modifications.¹⁹ (link) In brief, 1 ng of genomic DNA was fragmented in 5 µl of Amplicon Tagment Mix and 10 µl of Tagment DNA buffer. Tagmentation reaction was performed by incubation at 55 °C for 5 min followed by neutralisation with 5 µl of Neutralise Tagment Buffer for 5 min. Tagmented DNA (25 µl) was indexed in a 50 µl limited-cycle PCR (12 cycles) as outlined in the Nextera XT protocol and subsequently purified using 25 µl of AMPure XP beads (Beckman Coulter Inc, Australia). The fragment size distribution of the purified DNA was analysed utilising a 2100 Bioanalyser with a High Sensitivity DNA assay kit (Agilent Technologies, Santa Clara, CA). DNA libraries were adjusted to 2 nM, pooled in equal volumes and then denatured with 0.2 N NaOH according to the Nextera protocol. The libraries were sequenced using 2 × 300 paired-end protocols on an Illumina MiSeq instrument (MiSeq Reagent Kit v3 for 600 cycles).

Chua E.G., Wise M.J., Khosravi Y., Seow S.W., Amoyo A.A., Pettersson S., Peters F., Tay C.Y., Perkins T.T., Loke M.F., Marshall B.J, & Vadivelu J. (2016). Quantum changes in Helicobacter pylori gene expression accompany host-adaptation. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 24(1), 37-49.

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Plasma ctDNA Enrichment and Sequencing

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Libraries were prepared with Nextera Rapid Capture Custom Enrichment Kit (Illumina Inc., San Diego, CA, USA), according to the manufacturer’s protocol. Preparation of libraries was performed starting from up to 10–150 ng of plasma ctDNA and 50–100 ng of gDNA from PBMCs (as corresponding normal reference, or hg19 when germ-line DNA from the patient was not available). gDNA was fragmented using transposones, adding simultaneously adapter sequences. For ctDNA libraries preparation was used NEBNext^® Ultra™ DNA Library Prep Kit for Illumina^® (New England BioLabs Inc., Ipswich MA). Purified tagmented gDNA and ctDNA was used as template for subsequent PCR to introduce unique sample barcodes. Fragments’ size distribution of the DNA was assessed using the 2100 Bioanalyzer with a High Sensitivity DNA assay kit (Agilent Technologies, Santa Clara, CA). Equal amount of DNA libraries were pooled and subjected to targeted panel hybridization capture. Libraries were then sequenced using Illumina MiSeq or NextSeq500 sequencer (Illumina Inc., San Diego, CA, USA).

Siravegna G., Mussolin B., Buscarino M., Corti G., Cassingena A., Crisafulli G., Ponzetti A., Cremolini C., Amatu A., Lauricella C., Lamba S., Hobor S., Avallone A., Valtorta E., Rospo G., Medico E., Motta V., Antoniotti C., Tatangelo F., Bellosillo B., Veronese S., Budillon A., Montagut C., Racca P., Marsoni S., Falcone A., Corcoran R.B., Di Nicolantonio F., Loupakis F., Siena S., Sartore-Bianchi A, & Bardelli A. (2015). Monitoring clonal evolution and resistance to EGFR blockade in the blood of metastatic colorectal cancer patients. Nature medicine, 21(7), 795-801.

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Bisulfite Conversion and Sequencing of hCMV-MIE DNA

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Genomic DNA was extracted from recombinant CHO pools and treated with bisulphite as described previously12 (link). Bisulphite-converted hCMV-MIE DNA was amplified by PCR using primers 5′-GATATTGATTATTGATTAGTTATTAATAGTAATTAA-3′ and 5′-CAAATAAAAAAATCCCATAAAATCATATACTAA-3′ for amplicon 1 and primers 5′-TTAGTATATGATTTTATGGGATTTTTTTATTTG-3′ and 5′-TTCTAATACTAAACTCCTCTCCCAA-3′ for amplicon 2. Both amplicons were sequenced with the MiSeq system (Illumina, Inc., San Diego, USA). The concentration of amplified DNA was measured with the Qubit system (Life Technologies GmbH, Darmstadt, Germany). The library was generated according to the TruSeq Nano DNA Library Prep Guide (Part # 15041110 Rev. C, Illumina, Inc., San Diego, USA) using 100 ng of each amplified product. Library quality was checked by employing the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) and the High Sensitivity DNA Assay Kit (Agilent Technologies, Waldbronn, Germany). After verification of quality, 56 amplicons from different cell pools were pooled in equal shares and mixed with 10% PhiX before being run. Reads were mapped employing the Bismark software32 (link) which uses Bowtie233 (link) for alignment.

Moritz B., Becker P.B, & Göpfert U. (2015). CMV promoter mutants with a reduced propensity to productivity loss in CHO cells. Scientific Reports, 5, 16952.

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RNA-seq Library Preparation Protocol

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Total RNA was extracted from the specimens using TRIzol (Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions. The RNA was qualified and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). After rRNAs were removed, the rest of RNAs were fragmented using a fragmentation buffer and reverse-transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP), and buffer. Next, the cDNA fragments were purified using a QIAquick PCR Extraction Kit (Qiagen, Hilden, Germany), end-repaired, polyadenylated, and ligated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. After PCR amplification, the digested products were purified using AMPure XP Beads and a High Sensitivity DNA Assay Kit (Agilent Technologies, United States) was used for library quality inspection. RNA sequencing was carried out by Gene Denovo Biotechnology Company (Guangzhou, China) using a HiSeq 2500 system (Illumina, San Diego, CA, United States).

Li S.Y., Wang C.Y., Xiao Y.X., Tang X.B., Yuan Z.W, & Bai Y.Z. (2021). RNA-Seq Profiling of Circular RNAs During Development of Hindgut in Rat Embryos With Ethylenethiourea-Induced Anorectal Malformations. Frontiers in Genetics, 12, 605015.

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Bacterial DNA Extraction and Sequencing

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After bacterial culture, colonies were resuspended in 500 μL of water and bacterial pellets were digested using MagNA Pure 96 DNA Bacterial Lysis Buffer and proteinase K. DNA extraction was performed on a MagNA Pure 96 System (Roche Diagnostics, Penzberg, Germany) using the MagNA Pure 96 DNA and Viral NA SV Kit. Quantification and purity checks (260/280 and 260/230 ratios) were performed using NanoDrop (Thermo Scientific, Waltham, MA, United States) before external sequencing by Helixio (Saint-Beauzire, France³). Qubit quantification was carried out prior to sequencing. Library preparations were made using 1 ng of DNA and the Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, CA, United States) and validation of the libraries was performed on a bioanalyzer with the High Sensitivity DNA Assay kit (Agilent, Santa Clara, CA, United States) in order to obtain sizes ranging from 250 to 1,500 base pairs (bp). Paired-end sequencing was then performed on a NextSeq500 (Illumina). Quality was controlled using FastQC v0.11.3 (Wingett and Andrews, 2018 (link)). De novo assemblies were produced using SPAdes v3.10.1 (Bankevich et al., 2012 (link)).

Berthenet E., Bénéjat L., Ménard A., Varon C., Lacomme S., Gontier E., Raymond J., Boussaba O., Toulza O., Ducournau A., Buissonnière A., Giese A., Megraud F., Bessède E., Jehanne Q, & Lehours P. (2019). Whole-Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species. Frontiers in Microbiology, 10, 2820.

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