Qubit dsdna hs assay kit

Manufactured by Thermo Fisher Scientific

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The Qubit dsDNA HS Assay Kit is a fluorescence-based method for quantifying double-stranded DNA (dsDNA) in a sample. The kit provides a sensitive and accurate way to measure DNA concentration in a microplate or cuvette format.

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Lab products found in correlation

Qubit 2.0 fluorometer, by Thermo Fisher Scientific (598 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (494 mentions) Qubit fluorometer, by Thermo Fisher Scientific (307 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (227 mentions) Ampure xp bead, by Beckman Coulter (149 mentions) Qiaamp circulating nucleic acid kit, by Qiagen (129 mentions) 2100 bioanalyzer, by Agilent Technologies (120 mentions) Miseq platform, by Illumina (118 mentions) Nextseq 500, by Illumina (112 mentions) Nanodrop 2000, by Thermo Fisher Scientific (109 mentions) Qiaamp dna ffpe tissue kit, by Qiagen (109 mentions) Agilent high sensitivity dna kit, by Agilent Technologies (107 mentions) Agencourt ampure xp bead, by Beckman Coulter (103 mentions) Dneasy blood tissue kit, by Qiagen (100 mentions) Qubit 4 fluorometer, by Thermo Fisher Scientific (96 mentions) Qiaamp dna mini kit, by Qiagen (93 mentions) Nextera xt dna library preparation kit, by Illumina (92 mentions) Nanodrop, by Thermo Fisher Scientific (92 mentions) Dneasy blood and tissue kit, by Qiagen (86 mentions) Qubit 4.0 fluorometer, by Thermo Fisher Scientific (86 mentions)

Close spelling variants of this product

Qubit dsDNA HS Qubit dsDNA Assay Kit Qubit dsDNA assay DsDNA HS assay DsDNA Qubit Kit HS dsDNA kit Qubit HS kit DsDNA Qubit DsDNA assay kits Qubit assay

2 470 protocols using qubit dsdna hs assay kit

Comparative DNA and RNA Extraction Protocols

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First, we compared two DNA extraction kits: the QIAamp DNA Microbiome kit (Qiagen, Hilden, Germany) and the QIAamp UCP Pathogen Mini kit (Qiagen) for efficacy of detection of bacterial reads. Based on the results, the QIAamp DNA Microbiome kit was used in further experiments. RNA extraction was performed using the NucleoSpin RNA Blood kit (MACHEREY-NAGEL, Düren, Germany). The extracted RNA was immediately converted to cDNA and amplified with the REPLI-g WTA Single Cell kit (Qiagen) in accordance with the manufacturer’s instructions. DNA and RNA concentrations were measured using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit RNA HS assay kit (Thermo Fisher Scientific), respectively.
The Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) was used to prepare all the libraries from extracted DNA or generated cDNA, as described above. Libraries were also prepared from distilled water as a preparation control (NTC 1-3). Library quality was analyzed using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) or Agilent 2100 Bioanalyzer (Agilent Technologies). The library concentration was quantified using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific). Then, libraries were sequenced on MiSeq (Illumina) with the 2 × 150 bp paired-end protocol.

Takeuchi S., Kawada J.I., Horiba K., Okuno Y., Okumura T., Suzuki T., Torii Y., Kawabe S., Wada S., Ikeyama T, & Ito Y. (2019). Metagenomic analysis using next-generation sequencing of pathogens in bronchoalveolar lavage fluid from pediatric patients with respiratory failure. Scientific Reports, 9, 12909.

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DNA Sequencing Library Preparation

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DNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). 250 ng DNA was sheared in the Covaris® S2 instrument (Covaris, Inc.) to an insert size of approximately 650 bp. Fifty nanograms of sheared DNA was used for preparation of sequencing libraries with the ThruPLEX® DNA-seq Kit (Rubicon Genomics) according to the manufacturer’s instructions using provided primers with ten cycles for amplification. Primer sequences were as follows: 5’: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT with NNNNNNNN being a TruSeq HT i5 index and 3’: GTTCGTCTTCTGCCGTATGCTCTANNNNNNNNCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA with NNNNNNNN being a TruSeq HT i7 index.
PCR products were purified on the MBS Magnatrix 1200 automated workstation (NorDiag) with Dynabeads® MyOne carboxylic acid beads (Thermo Fisher Scientific). Purity of the samples and insert size distribution were inspected using the High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer instrument (Agilent Technologies). DNA libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and equimolar concentrations of 12 samples were pooled and further purified using Agencourt AMPure XP (Beckman Coulter, Inc.). Library pools with a final concentration of 10 nM DNA were submitted to one flow cell per pool and sequenced using the MiSeq V3 chemistry (Illumina).

Lindefeldt M., Eng A., Darban H., Bjerkner A., Zetterström C.K., Allander T., Andersson B., Borenstein E., Dahlin M, & Prast-Nielsen S. (2019). The ketogenic diet influences taxonomic and functional composition of the gut microbiota in children with severe epilepsy. NPJ Biofilms and Microbiomes, 5, 5.

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Whole Genome Sequencing of mcr-Positive Isolates

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Twenty-four mcr-positive isolates were characterized by WGS. Genomic DNA was extracted using DNeasy Blood & Tissue Kit and QIAcube Connect Device (QIAGEN, Hilden, Germany). For short-read sequencing, library preparation was performed using Illumina DNA prep kit (Illumina, San Diego, CA, USA) according to manufacturer instructions. Capillary electrophoresis using the Agilent 2100 Bioanalyzer system and Agilent High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA) was used for estimation of library quality. Concentration of the libraries was measured using the Qubit 4.0 fluorimeter with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed on the Illumina NextSeq 550 System with the NextSeq 500/550 Mid Output Kit v2.5 (300 Cycles) and on the Illumina MiSeq System with MiSeq Reagent Kit v2 (500-cycles) (Illumina, USA). For long-read sequencing, library preparation was performed with NEBNext Companion Module (New England Biolabs, Ipswich, MA, USA) and Ligation Sequencing Kit (Oxford Nanopore Technologies (ONT), Oxford, UK) in accordance with manufacturer’s protocol. Samples were multiplexed using Native Barcoding Expansion 1–12 (ONT, UK). Library concentration was measured with Qubit dsDNA HS Assay Kit and Qubit 4 Fluorometer (Thermo Fisher Scientific, USA). Sequencing was performed on the MinION platform and R9.4.1 Flow Cells (ONT, UK).

Shapovalova V., Shaidullina E., Azizov I., Sheck E., Martinovich A., Dyachkova M., Matsvay A., Savochkina Y., Khafizov K., Kozlov R., Shipulin G, & Edelstein M. (2022). Molecular Epidemiology of mcr-1-Positive Escherichia coli and Klebsiella pneumoniae Isolates: Results from Russian Sentinel Surveillance (2013–2018). Microorganisms, 10(10), 2034.

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Targeted DNA Sequencing for Mutational Profiling

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DNA was extracted from FFPE primary tumor biopsies using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA quality was evaluated based on the absorbance ratios of A260/280 and A260/230 using a NanoDropTM 2000c Spectrophotometer (Thermo Fisher, MA, USA). DNA quantity was determined using the Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). Two independent Targeted DNA sequencing panels were employed to allow the mutational profiling of 72 cancer driver genes (see Supplementary Data 1). DNA libraries that were built with GeneRead DNAseq Colorectal Cancer Panel V2 were processed and analyzed as was previously described (31 (link)). Of note, DNA libraries that were constructed with the AmpliSeqTM for Illumina Cancer Hotspot Panel v2 Kit that allow the detection of 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes, were prepared with 100 ng of genomic DNA as was previously described (40 (link)). These DNA libraries were measured using Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). All libraries were above the minimum concentration requirement of 2 nM for further sequencing in an Illumina MiSeq platform.

Iseas S., Sendoya J.M., Robbio J., Coraglio M., Kujaruk M., Mikolaitis V., Rizzolo M., Cabanne A., Ruiz G., Salanova R., Gualdrini U., Méndez G., Antelo M., Carballido M., Rotondaro C., Viglino J., Eleta M., Di Sibio A., Podhajcer O.L., Roca E., Llera A.S., Golubicki M, & Abba M.C. (2022). Prognostic Impact of An Integrative Landscape of Clinical, Immune, and Molecular Features in Non-Metastatic Rectal Cancer. Frontiers in Oncology, 11, 801880.

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SARS-CoV-2 Genome Sequencing from Clinical Samples

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Complementary DNA (cDNA) was prepared from the extracted RNA using SuperScript™ IV VILO™ (SSIV VILO) Master Mix (ThermoFisher Scientific MA USA). The cDNA was subjected to a multiplex PCR using the ARTIC nCoV-2019/V1 (first batch) or V3 (second batch) primers as per the protocol²⁰ (Josh Quick, 2020). The PCR products were visualized for the presence of 400 bp fragments and then purified using 1× Agencourt AMPure XP (Beckman Coulter Inc., TX USA). Products were quantified using the Qubit™ dsDNA HS Assay Kit (ThermoFisher Scientific, MA USA) and concentrations normalized to 1 ng. Libraries for sequencing were prepared from the normalized products using the Nextera XT DNA Library Preparation Kit and the Nextera XT Index Kit v2 Set-A (Illumina) according to manufacturer’s instructions. Each barcoded library was then purified using Agencourt AMPure XP beads (Beckman Coulter Inc., TX USA) and thereafter the size distribution and library quality control carried out using the Agilent 2100 Bioanalyzer. The purified libraries were quantified using the Qubit™ dsDNA HS Assay Kit on the Qubit 4.0 fluorometer (ThermoFisher Scientific, MA USA) and normalized to equimolar concentrations. A pool of all the normalized libraries was prepared and diluted to a final concentration of 10pM, spiked with 5% PhiX, and sequenced on the Illumina MiSeq system using the MiSeq® Reagent Kit v3 600 cycle.

Ngoi J.M., Quashie P.K., Morang'a C.M., Bonney J.H., Amuzu D.S., Kumordjie S., Asante I.A., Bonney E.Y., Eshun M., Boatemaa L., Magnusen V., Kotey E.N., Ndam N.T., Tei-Maya F., Arjarquah A.K., Obodai E., Otchere I.D., Bediako Y., Mutungi J.K., Amenga-Etego L.N., Odoom J.K., Anang A.K., Kyei G.B., Adu B., Ampofo W.K, & Awandare G.A. (2020). Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana. Experimental Biology and Medicine, 246(8), 960-970.

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DNA Extraction from Blood and Tumor Samples

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Peripheral blood samples were collected and centrifuged to separate buffy coats and plasma. Buffy coats were stored at − 80 °C until DNA extraction. Buffy coat DNA was extracted with the QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Hilden, Germany). The DNA concentration of buffy coats was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Plasma DNA was extracted with the MagMax Cell-Free DNA extraction kit on the KingFisher Duo Prime (Thermo Fisher Scientific). Concentration of plasma DNA was determined using the Qubit dsDNA HS Assay Kit and Qubit 3.0 fluorometer (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Tumor tissues were fixed using 10% buffered formalin^{29 (link)}. Serial 10 μm sections were prepared from formalin-fixed paraffin-embedded (FFPE) tissues, and sections were stained with hematoxylin–eosin and reviewed by a pathologist to check the tumor area. Laser capture microdissection was performed using an Arcturus XT laser microdissection system (Thermo Fisher Scientific). FFPE DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen), the GeneRead DNA FFPE Kit (Qiagen), and the MagMAX™ FFPE DNA/RNA Ultra Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. FFPE DNA concentrations were determined using the Qubit® dsDNA HS Assay Kit on a Qubit Fluorometer 3.0 (Thermo Fisher Scientific).

Hirotsu Y., Nakagomi T., Nagakubo Y., Goto T, & Omata M. (2024). Simulation analysis of EGFR mutation detection: Oncomine Dx target test and AmoyDx panel impact on lung cancer treatment decisions. Scientific Reports, 14, 1594.

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Shotgun Metagenomics of Rhizosphere Soils

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The rhizosphere soils collected were subjected to the DNA extraction protocol of the Qiagen DNeasy PowerMax Soil Kit (USA). Shotgun metagenomic sequencing was conducted on the extracted DNA at the Molecular Research LP, Texas, USA. Nextera DNA library preparation Flex kit (Illumina) was employed in the library preparation, following standard procedure. Evaluation of the initial DNA concentrations in the prepared samples was carried out using the Life Technologies Qubit® dsDNA HS Assay Kit produced by Carlsbad, USA. The samples were further cleaned with DNEasy PowerClean Pro Cleanup Kit (Qiagen) and the concentrations of the samples after the cleanup were again checked using the Qubit® dsDNA HS Assay Kit (Life Technologies, the USA). The library was then prepared using 20–25 ng of DNA, after which the samples were concurrently fragmented and an adapter sequence added. After that, the adapters were employed in a limited-cycle PCR to introduce unique indices into each sample. The final library concentrations were determined using the Qubit® dsDNA HS Assay Kit (Life Technologies) after the libraries were prepared, and the average size of the library was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries were pooled and diluted to 0.6 nM before being sequenced for 300 cycles paired-end on the NovaSeq system (Illumina) 6000 system.

Dlamini S.P., Akanmu A.O., Fadiji A.E, & Babalola O.O. (2022). Maize rhizosphere modulates the microbiome diversity and community structure to enhance plant health. Saudi Journal of Biological Sciences, 30(1), 103499.

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Bacterial 16S rDNA Sequencing via PacBio SMRT

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The genomic DNA was tested for quality and purity, and then sequencing was performed using a PacBio SMRT system. The concentration of the extracted DNA was verified again with a Qubit dsDNA HS Assay Kit (Thermo Scientific). The DNA quality was verified by 1% agarose gel electrophoresis. After the second verification reached the standard, PCR amplification was performed. In addition, PCR reactions were conducted with the bacterial universal primers 27-Forward (barcode: AGRGTTYGATYMTGGCTCAG) and 1492-Reverse (barcode: RGYTACCTTGTTACGACTT) so as to amplify the full length of the 16S rDNA gene, whereas PCR amplification was carried out as described by She et al. (2018) (link). The total PCR amplicons were purified with Agencourt AMPure XP Beads (Beckman Coulter) and quantified using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). After the individual quantification step, amplicons were pooled in equal amounts. The SMRT bell libraries were prepared from the amplified DNA by SMRT bell Express Template Prep Kit 2.0 according to the manufacturer's instructions (Pacific Biosciences). Purified SMRT bell libraries from the pooled and barcoded samples were sequenced on a single PacBio Sequel II 8M cell using the Sequel II Sequencing kit 2.0.

Liang L., Wang P., Zhao X., He L., Qu T, & Chen Y. (2022). Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China. Journal of dairy science, 105(7).

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Fecal DNA Extraction and Microbiome Analysis

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DNAs were isolated from fecal using the QIAamp DNA Mini Kit (Qiagen, 51306). The concentration of double-stranded DNA in isolated samples was determined using a Qubit 2.0 instrument and a Qubit dsDNA HS Assay kit (ThermoFisher, catalog number 32853).
Library for Next-generation sequencing (NGS) generated with NEXTflex® 16S V1-V3 Amplicon-Seq Kit (PerkinElmer, catalog number NOVA-4202-04), according to the manufacturer's instructions. The library quality was quantified by Qubit dsDNA HS Assay kit with the Qubit 2.0 fluorometer system (Invitrogen, Life Technologies, Grand Island, NY, USA). Amplicons were sequenced on the MiSeq instrument (Illumina Systems).
Demultiplexing, filtering, denoise, chimeric sequences, and determining OTU and taxonomic identification were performed using LotuS pipeline (Hildebrand et al. 2014) (link).
Analysis of alpha diversity to assess the abundance of the community, the calculation of alpha biodiversity (Shannon indices), beta biodiversity as well as the construction of taxonomic distribution at the phylum and genus level were performed using vegan (Oksanen et al. 2019 ) and phyloseq R packages (v.1.24.2) (McMurdie and Holmes 2013) and graphs were generated using web-based platform for comprehensive analysis -MicrobiomeAnalyst (Chong et al. 2020) (link).

Kozhakhmetov S., Babenko D., Nurgaziyev M., Tuyakova A., Nurgozhina A., Muhanbetganov N., Chulenbayeva L., Sergazy S., Gulyaev A.E., Saliev T., & Kushugulova А. (2020). The combination of mare's milk and grape polyphenol extract for treatment of dysbiosis induced by dextran sulfate sodium. Biodiversitas Journal of Biological Diversity, 21(5).

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Metagenomics Sequencing Protocol for Soil

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Total DNA was extracted as per the manufacturer’s protocol using FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). Libraries for metagenomic sequencing were prepared according to the manufacturer’s protocol using Nextera XT Library Preparation Kit (Illumina #FC-131-1096). Library preparation and beads clean-up were run on the Mantis Liquid Handler (Formulatrix) and Epimotion (Eppendorf #5075000301) automated platform. Libraries were quantified and quality control was performed using the QubitTM dsDNA HS Assay Kit (Invitrogen) and Agilent D5000 HS tapes (#5067-5592) on the TapeStation 4200 (Agilent #G2991AA). The sequencing pool was created with equimolar amounts of 1 nM per library and quantified in triplicates using the QubitTM dsDNA HS Assay Kit (Invitrogen). The libraries were sequenced on the NextSeq500 (Illumina, USA) using NextSeq 500/550 High Output v2 2x150bp paired-end chemistry at the Australian Centre for Ecogenomics (ACE). Generated reads were quality-checked using FastQC-v0.11.7 (Simon Andrews, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Duplicates were removed using FastUniq-1.1 [15 (link)], and the remaining reads were trimmed using Trimmomatic 0.36 [16 (link)]. Reads shorter than 100 bp were discarded.

Li J., Liu T., McIlroy S.J., Tyson G.W, & Guo J. (2023). Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings. ISME Communications, 3, 39.

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