A randomly chosen subcollection of the isolates tested for susceptibility (n = 96) were analyzed by whole-genome sequencing (WGS). One colony of each isolate was incubated in 2 ml Luria-Bertani broth for 8 h at 37°C. DNA was prepared by use of a Wizard Genomic DNA purification kit (Promega, USA) according to the manufacturer's recommendations for Gram-negative bacteria, with the exception that DNA was rehydrated with 10 mM Tris-HCl (pH 8.0). The quality and quantity of the extracted DNA were assessed by gel electrophoresis, spectrophotometry (NanoDrop; Thermo Fisher), and a Quant-iT dsDNA BR assay with a Qubit instrument (Invitrogen, USA). After standardization of the DNA extracts, the samples were transferred to the Oxford Genome Center for library preparation and WGS. Briefly, fragmented DNAs were end repaired, A-tailed, adapter ligated, and amplified using a Nextera DNA library prep kit (Illumina, USA). Sequencing was done on an Illumina HiSeq4000 platform, generating 150-bp paired-end reads.
Quant it dsdna br assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Quant-iT dsDNA BR assay is a fluorescence-based method for quantifying double-stranded DNA (dsDNA) in solution. It utilizes a DNA-binding dye that emits fluorescence upon binding to dsDNA. The fluorescence intensity is directly proportional to the dsDNA concentration, allowing for accurate quantification over a wide range of concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Qubit instrument, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Nextera dna library prep kit, by Illumina (1 mentions)
Agilent bioanalyzer 2100 dna 1000 chip, by Agilent Technologies (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Allprep powerviral dna rna kit, by Qiagen (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Nextera dna library prep, by Illumina (1 mentions)
Lb broth, by Merck Group (1 mentions)
Truseq dna lt sample prep kit, by Illumina (1 mentions)
Mate pair library prep kit v2, by Illumina (1 mentions)
6 protocols using quant it dsdna br assay
1
Whole-Genome Sequencing of Bacterial Isolates
2
Cell Proliferation Quantification by DNA
3
Microbiome Analysis of Swab Samples
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DNA extraction was performed using the Allprep PowerViral DNA/RNA Kit (Qiagen, Venlo, the Netherlands) with modifications. Briefly, swabs were suspended in 100 μL PowerBead solution and spun in a centrifuge at 24,000 RPM for five minutes. Swabs were then removed and the resulting eluant was supplemented with an additional 500 μL of PowerBead solution. The remainder of protocol was carried out per the manufacturers’ protocol. All samples produced low DNA yields (<2 ng/uL) as measured by Quant-iT dsDNA BR assay (Invitrogen, Waltham, MA, USA). Library preparation and sequencing was attempted on all samples with experimental and corresponding negative control samples processed concurrently.
The V3-V4 hypervariable region of the 16S rRNA gene was amplified using previously described primers and protocol.21 (link) Concentration and purity of PCR products were determined by PicoGreen (Invitrogen, Carlsbad, CA, USA) and an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Santa Clara, CA), respectively. Amplicons were pooled in equimolar concentrations and sequencing performed on the Illumina MiSeq and the MiSeq reagent kit v3 (600-cycle) according to the manufacturers’ instructions.
The V3-V4 hypervariable region of the 16S rRNA gene was amplified using previously described primers and protocol.21 (link) Concentration and purity of PCR products were determined by PicoGreen (Invitrogen, Carlsbad, CA, USA) and an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Santa Clara, CA), respectively. Amplicons were pooled in equimolar concentrations and sequencing performed on the Illumina MiSeq and the MiSeq reagent kit v3 (600-cycle) according to the manufacturers’ instructions.
4
Whole-Genome Sequencing of Bacteria
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One colony of each isolate was incubated in 2 mL LB broth (Sigma-Aldrich, USA) for 8 h at 37°C in room atmosphere. DNA preparation was done using a Wizard ® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's recommendations for Gram-negative bacteria with the exception that DNA was rehydrated with 10 mM Tris-HCl (pH 8.0). The quality and quantity of the extracted DNA was controlled by gel electrophoresis, spectrophotometry (Nanodrop, Thermo Fisher), and Quant-iT dsDNA BR assay and a Qubit instrument (Invitrogen). After standardizing the DNA extracts, the samples were transferred to Oxford Genome Center for library preparation and whole-genome sequencing. Fragmented DNA was end-repaired, A-tailed, adapter-ligated, and amplified using Nextera DNA library Prep (Illumina, USA). Sequencing was done on an Illumina HiSeq4000 platform, generating 150 bp paired-end reads.
5
DNA Concentration Quantification using Qubit
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The concentration of DNA obtained after the different extraction procedures for each sample was quantified using a Qubit 3.0 fluorometer with the Quant-iT dsDNA HS Assay (assay range between 0.2 and100 ng; sample concentration between 10 pg/μl and 100 ng/μl) and the Quant-iT dsDNA BR Assay (assay range between 2 and 1000 ng; sample concentration between 100 pg/μl and 1000 μg/μl), according to manufacturer's instructions (ThermoFischer Scientific).
6
De novo Genome Assembly of P. agarexedens
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The genomic DNA of P. agarexedens was isolated and quantified using a Quant-iT dsDNA BR assay (Thermo Fisher Scientific). The quality of the extracted genomic DNA was verified on a 0.6% agarose gel. DNA libraries were constructed using an Illumina TruSeq DNA LT Sample Prep Kit. Mate-pair libraries were constructed using an Illumina Mate Pair Library Prep Kit v2. For the de novo assembly of the complete genome, we used Velvet (Zerbino and Birney 2008 (link)) to assemble paired-end and mate-pair reads to scaffolds. Genes on these assembled scaffolds were predicted using GeneMark.hmm (Besemer and Borodovsky 1999 (link)) and were annotated using a BLAST (blastp) search against the NCBI nr protein database, with an e-value cutoff of 0.00001. For the functional annotation of predicted genes, the accession numbers from BLAST hits were mapped to GO terms by querying the GO database (Ashburner et al. 2000 (link)). Enzyme code annotations were retrieved by mapping to GO terms, and enzyme codes were retrieved by querying the GO database.
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