Total DNA was isolated from 250 mg of ileal samples (n = 57) and cecal samples (n = 57) using a PowerSoil DNA isolation kit (MoBio Laboratories Inc., Carlsbad, CA, USA) with modifications performed as previously described (30 (link)). The DNA concentration was quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and a Qubit double-stranded DNA (dsDNA) HS assay kit (Life Technologies). An aliquot of each of the DNA samples was sent to a commercial provider (Microsynth AG, Balgach, Switzerland). The V3-V5 hypervariable region of the 16S rRNA gene was amplified using primers 357F-HMP (5′-CCTACGGGAGGCAGCAG-3′) and 926R-HMP (5′-CGTCAATTCMTTTRAGT-3′) to generate an approximate amplicon size of 570 bp (31 (link)) and a Kapa HiFi HotStart PCR kit (Roche, Baden, Switzerland), which included high-fidelity DNA polymerase. Libraries were constructed by ligating sequencing adapters and indices onto purified PCR products using a Nextera XT sample preparation kit (Illumina Inc., San Diego, CA, USA) and the recommendations of the manufacturer. Equimolar amounts for each library were pooled and sequenced on an Illumina MiSeq Personal Sequencer using a 300-bp read length paired-end protocol. Afterwards, FASTQ files were demultiplexed, quality filtered, and trimmed of Illumina adaptor residuals, and the overlapping paired-end reads were stitched by Microsynth.
Qubit double stranded dna dsdna hs assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit double-stranded DNA (dsDNA) HS assay kit is a fluorescence-based method for quantifying dsDNA. The kit provides reagents and standards for accurately measuring dsDNA concentrations from 0.2 to 100 ng/mL.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Nextera xt kit, by Illumina (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Miseq system, by Illumina (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Exosap it pcr product cleanup reagent, by Thermo Fisher Scientific (2 mentions)
Miseq reagent micro kit v2, by Illumina (2 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Nextera xt sample preparation kit, by Illumina (1 mentions)
Kapa hifi hotstart pcr kit, by Roche (1 mentions)
Miseq personal sequencer, by Illumina (1 mentions)
Mh broth, by BD (1 mentions)
Qubit fluorimeter 2, by Thermo Fisher Scientific (1 mentions)
Nextera xt index kit v2, by Illumina (1 mentions)
Masterpure complete dna and rna purification kit, by Illumina (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
17 protocols using qubit double stranded dna dsdna hs assay kit
1
16S rRNA Gene Sequencing of Ileal and Cecal Samples
2
Whole Genome Sequencing of Resistant Bacterial Isolates
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MH broths were inoculated with phenotypically confirmed ESBL-E, CPE, and VRE isolates and cultured in Muller–Hinton (MH) broth (BD, Erembodegem, Belgium) for 18 to 24 h. DNA was isolated from 500 μl of the broth culture using the MasterPure™ Complete DNA and RNA Purification Kit (Epicenter, Madison, USA). DNA concentration was measured with a Qubit Fluorimeter 2.0 (ThermoFisher Scientific, Waltham, USA) using the Qubit Double-Stranded DNA (dsDNA) HS Assay Kit (Life Technologies, Carlsbad, USA). A concentration of 0.24–0.30 ng/μl of bacterial DNA was used for library preparation using the Nextera XT Library Preparation Kit with the Nextera XT v2 Index Kit (Illumina, San Diego, USA), according to the manufacturer’s instructions. Sequencing of the library was performed on a MiSeq sequencer, using the MiSeq Reagent Kit v2 generating 250-bp paired-end reads. A harmonisation study for WGS was performed within the i-4-1-Health project [16 (link)].
3
DNA Extraction from Nasonia vitripennis
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Some of the following methods are similar to those previously published (31 (link)). The founders for each generation of N. vitripennis were collected and maintained at −80°C until DNA and RNA extraction. For DNA extraction, each individual was put in a 1.5-ml tube and rinsed once with 1 ml of 70% EtOH and 1 ml of 10% bleach and twice with 1 ml of sterile water. Samples were then frozen and homogenized in liquid nitrogen. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was quantified using the Qubit double-stranded DNA (dsDNA) HS assay kit on the Qubit 2.0 fluorometer (Life Technologies).
4
Extraction and Amplification of Genomic DNA
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Genomic DNA was extracted using the MagMAX Total nucleic acid isolation kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s protocol with the repeat bead beating method on a FastPrep-24 instrument (MP Biomedicals LLC). The DNA concentration was measured with the Qubit 3.0 fluorometer using the Qubit double-stranded DNA (dsDNA) HS assay kit (Life Technologies, Eugene, OR). PCR amplification was performed using Ex Taq DNA polymerase (TaKaRa, Otsu, Japan) and primers F515 and R806 (50 (link)) with a random 8-bp barcode on the 5ʹ end of F515 for sample multiplexing (51 (link), 52 (link)). PCR was initiated at 94°C for 3 min, followed by 35 cycles of 94°C for 45 s, 54°C for 60 s, and 72°C for 30 s, with a final extension step at 72°C for 10 min. Negative controls were run for each barcoded primer. No PCR product for the negative controls was observed on a 1.5% agarose gel. PCR products were pooled and then gel purified with the Wizard SV gel and PCR clean-up system (Promega, Madison, WI).
5
Plasmodium Genome DNA Isolation and Sequencing
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Plasmodium genome DNA was isolated using the genome DNA isolation kit. DNA concentrations were measured on the Qubit double-stranded DNA (dsDNA) HS assay kit (Invitrogen). Libraries for paired-end sequencing were constructed from DNA extracts ranging from <50 ng/ml to 0.2 ng/μl, using the Illumina NexteraXT kit (FC-131-1024, Illumina, CA, USA). The pooled NexteraXT libraries were loaded onto an Illumina NextSeq 550 system in house at IISER, Pune with a standard flow cell.
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Plasmodium genome DNA was isolated using the genome DNA isolation kit. DNA concentrations were measured on the Qubit double-stranded DNA (dsDNA) HS assay kit (Invitrogen). Libraries for paired-end sequencing were constructed from DNA extracts ranging from <50 ng/ml to 0.2 ng/μl, using the Illumina NexteraXT kit (FC-131-1024, Illumina, CA, USA). The pooled NexteraXT libraries were loaded onto an Illumina NextSeq 550 system in house at IISER, Pune with a standard flow cell.
6
Illumina NexteraXT Library Preparation
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DNA concentrations in extracts were measured on the Qubit double-stranded DNA (dsDNA) HS assay kit (Invitrogen). Libraries for paired-end sequencing were constructed from DNA extracts ranging from < 50 ng/ml to 0.2 ng/µl, using the Illumina NexteraXT (Illumina, California, USA) Guide 150319425031942 and following protocol revision E. The Pooled NexteraXT libraries were loaded onto an Illumina MiSeq reagent cartridge using MiSeq reagent kit v3 and 500 cycles with a standard flow cell.
7
Extracting DNA from FFPE Tissue
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DNA was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen, CA, USA) and was quantified by the Qubit double-stranded DNA (dsDNA) HS assay kit on the Qubit 3.0 fluorometer (Thermo Fisher Scientific, NH, USA) following the manufacturer’s instructions.
8
Amplification and Purification of MR4 DNA
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Amplification reactions were carried out with 5 ng of MR4 reference strain DNA or 2 μl of clinical isolate DNA as the template in a 25-μl reaction volume with Phusion high-fidelity PCR master mix, HF buffer (Thermo Fisher Scientific), and a 0.2 μM concentration of forward and reverse primers. The cycling parameters used to amplify all loci were as follows: 98°C for 30 s, 35 cycles of (98°C for 10 s and 64°C for 5 min 30 s) (35 (link)), with a final extension at 64°C for 10 min. Amplicons were visualized on a 1% agarose gel stained with ethidium bromide, using a GeneRuler 1-kb Plus DNA ladder (Thermo Fisher Scientific). PCR products from a single sample were pooled and purified using the QIAquick PCR purification kit (Qiagen, CA, USA) and quantified using a Qubit double-stranded DNA (dsDNA) HS assay kit (Thermo Fisher Scientific) on the Qubit 2.0 fluorometer (Thermo Fisher Scientific).
9
Comprehensive DNA Extraction Protocols
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Cancer samples were preprocessed for DNA extraction comparably to a recent study on pancreatic cancer microbiota (48 (link)), urine samples according to a recent publication on how to study urinary microbiota (32 (link)), and other samples according to in-house methods for sample processing (Table S1 ). For solid cancer samples, the beating steps during preprocessing were performed using a Qiagen TissueLyser LT (Qiagen Benelux, Venlo, The Netherlands) at 50 Hz for 1 min (Table S1 ). As single saliva samples did not contain sufficient volume for multiple extractions, several samples from the same individual were pooled to obtain the appropriate volume. DNA was extracted in duplicate from three unique samples for each biological material, only for oral swabs from six unique samples, and from the two mock communities. DNA was extracted using three different extraction protocols (see “DNA extraction protocols” below), and for each protocol a negative extraction (no sample) was included in duplicate. The DNA standard was taken along in duplicate. DNA was quantified using a Qubit 3.0 fluorometer (Invitrogen, Breda, The Netherlands) and the Qubit double-stranded DNA (dsDNA) HS assay kit (Thermo Fisher, Landsmeer, The Netherlands). A schematic overview of the study setup is shown in Fig. 1 .
10
Genomic DNA Extraction from Legionella pneumophila
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Genomic DNA was extracted from 10 L. pneumophila isolates using the QIAamp DNA blood mini kit (Qiagen) with a 3-h proteinase K digest at 56°C. The final DNA extract was eluted in DNase-/RNase-free water, and the quality was measured using the NanoDrop ND-1000 (NanoDrop Technologies, Thermo Fisher Scientific, Australia). DNA purity was deemed to be satisfactory at a 260-nm/280-nm ratio of 1.6 to 2.2 and a 260-nm/230-nm ratio of >1.8. DNA concentration was measured by using the Qubit double-stranded DNA (dsDNA) HS assay kit (Thermo Fisher Scientific, Australia).
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