The Invisorb Spin Plant Mini Kit (Stratec Molecular, GmbH,Berlin, Germany) was used to isolate genomic DNA and prepare short-read libraries for the Roche-454 and Illumina sequencing platforms. DNA concentrations were determined with the Quant-iT dsDNA Assay Kit (Life Technologies, Carlsbad, California, USA) and a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA). We checked the integrity of the genomic DNA by agarose gel electrophoresis and pulsed-field gel electrophoresis. Agarose-embedded high-molecular weight (HMW) DNA was prepared as described by Peterson et al.48 , and modified as described by Zhang et al.49 (link), to construct Illumina TruSeq Synthetic Long Read (TSLR) libraries. Agarose gel plugs were washed three times in TE buffer, and subjected to digestion with 8 U of β-agarase (New England Biolabs, Ipswich, Massachusetts, USA) for 12-16 hours at 42°C. HMW DNA was then drop-dialyzed for 2.5 hours. DNA concentrations were quantified with the Quant-iT dsDNA Assay Kit (Life Technologies). DNA quality was then checked with Argus™ Qcard kit (OpGen, Maryland, USA) and was estimated at 20 to 100 kb.
Quant it dsdna assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The Quant-iT dsDNA Assay Kit is a fluorescence-based detection system designed for the quantification of double-stranded DNA (dsDNA) samples. The kit provides a sensitive and accurate method for measuring dsDNA concentrations in a microplate format.
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Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (11 mentions)
Gelase, by Illumina (8 mentions)
Agencourt ampure xp, by Beckman Coulter (7 mentions)
Miseq platform, by Illumina (6 mentions)
Large dna purification kit, by Bio-Rad (4 mentions)
Miseq reagent kit v2, by Illumina (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Dneasy powersoil htp 96 dna isolation kit, by Qiagen (3 mentions)
Fastprep 24 bead beater, by MP Biomedicals (3 mentions)
Dneasy powersoil kit, by Qiagen (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Hotstarttaq plus master mix kit, by Qiagen (3 mentions)
Miseq paired end 2, by Illumina (3 mentions)
Bp sequencing, by Illumina (3 mentions)
β agarase, by New England Biolabs (2 mentions)
Invisorb spin plant mini kit, by Stratec (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Plant dneasy, by Qiagen (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Amplification grade dnase 1, by Merck Group (2 mentions)
107 protocols using quant it dsdna assay kit
1
Genomic DNA Extraction and Sequencing Library Preparation
2
Targeted 16S rRNA Microbiome Sequencing
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DNA extraction from the samples, preparation of a 16S rRNA library, and sequencing were conducted at Takara Bio, Inc (Kusatsu, Japan) using the MiSeq system (Illumina, San Diego, Calif) according to the manufacturer's protocol. DNA was extracted from the samples using the NucleoSpin Soil kit (Macherey-Nagel, Düren, Germany). All extracted DNA samples were quantified by fluorescence detection using the Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, Mass) and purified using Agencourt AMPure XP (Beckman Coulter, Brea, Calif). Sequencing libraries were prepared using the 16S (V3-V4) Metagenomic Library Construction Kit for NGS (Takara Bio, Kusatsu, Japan). The first polymerase chain reaction amplification was performed using the primer pair 341 F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and 806 R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′) with Illumina adaptor overhang sequences. The second polymerase chain reaction amplification was performed using the Nextera XT Index Kit v2 (Illumina). Sequencing libraries were purified using Agencourt AMPure XP (Beckman Coulter) and quantified by fluorescence detection using the Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Clonal clusters of the libraries were generated and sequenced on a MiSeq system (Illumina) with the MiSeq Reagent v3 kit (Illumina) in the 2 × 250-bp mode.
3
Cell Proliferation on Polymer/α-TCP Scaffolds
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On days 1 and 3, the amount of cells on the scaffolds (cell proliferation) was determined by means of dsDNA quantification (Quant-iT™ dsDNA Assay Kit; Life Technologies, Carlsbad, CA, USA). The polymer/α-TCP samples seeded with cells were placed in 300 μL of cell lysis solution (0.2% v/v Triton X-100, 10 mM Tris (pH 7.0), and 1 mM EDTA) and processed through 3 freeze/thaw cycles (vortexed between each cycle). The samples were further processed according to the manufacturer’s instructions and measured using a multiplate fluorescence reader (Synergy HT, Winooski, VT, USA; λex = 485 nm, λem = 525 nm). The averaged dsDNA quantification values were determined after 24 and 72 h with respect to at least 5 independently prepared samples. The data were normalized to the values obtained after 24 h of cell culture.
4
Quantitative Cell Proliferation Assay
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Cell proliferation was measured using Quant-iT™ dsDNA Assay Kit (Life Technologies). The samples were washed twice with PBS and put into 500 μL lysate buffer (0.2% v/v Triton X-100, 10 mM Tris (pH 7.0) and 1 mM EDTA). The samples were frozen/thawed in three cycles (−80 °C/room temperature). The amount of dsDNA was evaluated according to the manufacturer's instructions using a multidetection reader Tecan Infinite® M200 Pro, λex = 485 nm, λem = 523 nm.
5
Stool DNA Extraction Protocol
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Stool was weighed, and DNA extracted using the DNeasy PowerSoil HTP 96 DNA Isolation kit (Qiagen, Chadstone VIC, Australia; Cat No. 12888–100). The following modification to the manufacturer’s instructions were employed: samples and solution C1 were added into bead tubes and heated for 10 min at 65 °C, prior to two cycles of bead beating at 6.5 m/s for 1 min using a FastPrep-24 bead beater (MP Biomedicals, Santa Ana, CA, USA). Quant-IT dsDNA Assay kit (Life Technologies, Carlsbad, CA, USA) was used to quantify DNA concentration after extraction. Extracted DNA was stored at − 20 °C prior to further analysis.
6
High-Molecular-Weight DNA Purification
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Mammalian cells were embedded in gel plugs and High Molecular Weight DNA was purified as described in a commercial large DNA purification kit (BioRad #170–3592). Plugs were incubated with lysis buffer and proteinase K for four hours at 50°C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to four hours of drop-dialysis. It was quantified using Quant-iTdsDNA Assay Kit (Life Technology), and the quality was assessed using pulsed-field gel electrophoresis.
7
Identification of F. psychrophilum via PCR
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Bacterial genomic DNA was extracted from 7‐day‐old bacterial cultures using the DNeasy Blood and Tissue kit (Qiagen Inc.) according to the manufacturer's protocol for Gram‐negative bacteria. Nucleic acids were then quantified using the Quant‐iT dsDNA Assay kit and a Qubit fluorometer (Life Technologies) and diluted to 20 ng/μl using nuclease‐free water (ThermoFisher Scientific). Yellow‐pigmented bacteria suspected of being F. psychrophilum were assayed using the F. psychrophilum‐specific endpoint PCR assay of Toyama et al. (1994 ) as previously described (Van Vliet et al., 2015 (link)). The template for negative control reactions consisted of nuclease‐free water, whereas positive control template was derived from a previously sequenced‐confirmed F. psychrophilum isolate. Resultant PCR products were electrophoresed in a 1.5% agarose gel for 30 min (100 V) and then visualized under UV transillumination. The presence of a ~ 1,088 base pair‐sized amplicon was considered confirmatory for bacterial identification as F. psychrophilum (Toyama et al., 1994 ).
8
Mammalian High Molecular Weight DNA Purification
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Mammalian cells were embedded in gel plugs and High Molecular Weight DNA was purified as described using a commercial large DNA purification kit (BioRad #170–3592). Plugs were incubated with lysis buffer and proteinase K for four hours at 50 °C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to 2.5 hours of drop-dialysis. It was quantified using the Quant-iT dsDNA Assay Kit (Life Technology), and the quality was assessed using pulsed-field gel electrophoresis43 (link).
9
Cell Metabolic Activity and DNA Measurement
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On Days 6 and 15, the metabolic activity of cells was measured using an MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA), according to the manufacturer’s manual. Lysis buffer was further added to the samples, and the amount of DNA was measured using the Quant-iT™ dsDNA Assay Kit (Life Technologies, Eugene, OR, USA) according to the manufacturer’s manual. The data were processed using the calibration curve of the standards from the kit.
10
Genomic DNA Isolation and Library Preparation
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An Invisorb Spin Plant Mini Kit (Stratec Molecular) was used to isolate genomic DNA and prepare short-read libraries for the Roche-454 and Illumina sequencing platforms. DNA concentrations were determined using a Quant-iT dsDNA Assay Kit (Life Technologies) and a Qubit Fluorometer (Invitrogen). We checked the integrity of the genomic DNA by agarose gel electrophoresis and pulsed-field gel electrophoresis. Agarose-embedded high-molecular weight (HMW) DNA was prepared as described previously48 , and modified as described previously49 (link), to construct Illumina TruSeq Synthetic Long Read (TSLR) libraries. Agarose gel plugs were washed three times in Tris EDTA buffer and subjected to digestion with 8 U of β-agarase (New England Biolabs) for 12–16 h at 42 °C. HMW DNA was then drop-dialysed for 2.5 h. DNA concentrations were quantified with the Quant-iT dsDNA Assay Kit. DNA quality was then checked using an Argus Qcard Kit (OpGen) and was estimated at 20–100 kb.
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