DNA was isolated from cell pellets with the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for tissue isolation. DNA concentration was measured using a Qubit™ 4 fluorometer (Invitrogen/Fisher Scientific, Schwerte, Germany). Qubit™ assay tubes and a Qubit™ 1× dsDNA HS Assay Kit (Invitrogen, Eugene, OR, USA) were used according to the manufacturer’s protocol.
Qubit assay tube
Manufactured by Thermo Fisher Scientific
Sourced in United States
Qubit assay tubes are specialized containers designed for use with the Qubit Fluorometer, a sensitive instrument used to accurately measure the concentration of DNA, RNA, and protein samples. These tubes are optimized to provide precise and reliable results when analyzing sample concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (2 mentions)
Thermomixer c 15, by Eppendorf (2 mentions)
Qubit 1 dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Mops sds running buffer 20x, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Quant it protein assay kit, by Thermo Fisher Scientific (1 mentions)
Maxwell rsc ccfdna plasma kit, by Promega (1 mentions)
Quantus fluorometer, by Thermo Fisher Scientific (1 mentions)
5424 microcentrifuge, by Eppendorf (1 mentions)
High sensitivity d1000 screentape, by Agilent Technologies (1 mentions)
Hiseq 4000 system, by Illumina (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
High sensitivity d1000 reagents, by Agilent Technologies (1 mentions)
Dynamag 2 magnetic rack, by Thermo Fisher Scientific (1 mentions)
Pippin prep dna size selection system, by Sage Science (1 mentions)
9 protocols using qubit assay tube
1
DNA Isolation and Quantification
2
Protein Quantification and Separation in EVs
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Protein concentrations in EVs samples were determined using the Qubit Protein Assay Kit (Thermo Fisher Scientific). Measuring was done using the Qubit Fluorometer (Thermo Fisher Scientific). This assay was performed at room temperature using specific Qubit assay tubes (Cat. no. Q32856).
For protein separation, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini Gel Tank electrophoresis system (Invitrogen). Proteins were denatured in a loading buffer (1X LDS sample buffer + β -mercaptoethanol) at 98 °C for 5 min. Samples were loaded and separated on a NuPAGE 4–12% Bis–Tris Gel (Invitrogen) using 1X MOPS SDS Running Buffer 20X (Life technologies) at a constant voltage of 110 V and for 1:30 min. After the run was completed, several bands were identified. Gel images were visualized in the ChemiDoc MP Imaging System (Bio-Rad).
For protein separation, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini Gel Tank electrophoresis system (Invitrogen). Proteins were denatured in a loading buffer (1X LDS sample buffer + β -mercaptoethanol) at 98 °C for 5 min. Samples were loaded and separated on a NuPAGE 4–12% Bis–Tris Gel (Invitrogen) using 1X MOPS SDS Running Buffer 20X (Life technologies) at a constant voltage of 110 V and for 1:30 min. After the run was completed, several bands were identified. Gel images were visualized in the ChemiDoc MP Imaging System (Bio-Rad).
3
Protein Quantitation Using Qubit Fluorometer
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The protein quantitation was carried out using a commercial supply (Quant-iT™ Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). The Quant-iT protein reagent was diluted with a ratio of 1:200 with Quant-iT protein buffer as a working solution. The protein standard solution was prepared by adding 10 μL of BSA standard to 190 μL of working solution to form a final analytic volume of 200 μL. Similarly, the sample solutions were prepared using 180–199 μL of working solution with 1–20 μL of unknown samples to form a final analytic volume of 200 μL. The standard solutions and sample solutions were loaded into the Qubit® assay tubes (Thermo Scientific, Waltham, MA, USA), the assay was performed at room temperature for 15 min, and then the fluorescence reading was measured using a Qubit® fluorometer. Duplicates of the standards and the unknown samples were performed.
4
ccfDNA Extraction from Plasma
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ccfDNA was extracted using the Maxwell RSC ccfDNA Plasma Kit (RSC; Promega, Leiden, The Netherlands). A total of 50 µL of ccfDNA were extracted from 1000 µL of plasma according to the manufacturer’s protocol. All the samples were quantified by Quantus Fluorometer and/or Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Aalst, Belgium) using Qubit assay tubes (cat. Q32856) and following the manufacturer’s protocol.
5
Sequencing Library Preparation Protocol
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1.7 mL microcentrifuge tubes (Denville, cat. no. C2170)
0.2 mL PCR 8-Strip tubes (Midsci, cat. no. AVSST)
Eppendorf Microcentrifuge 5424 (Eppendorf, cat. no. 5424 000.614)
Eppendorf fixed-angle rotor (Eppendorf, cat. no. 5424 702.007)
Digital Incublock (Denville, cat. no. I0520)
Modular block (Denville, cat. no. I9013)
Applied Biosystems Thermal Cycler 2720 (Life Technologies, cat. no. 4359659)
NanoDrop™ 8000 Spectrophotometer (ThermoFisher Scientific, cat. no. ND-8000-GL)
Electrophoresis gel system (USA Scientific, cat. no. 3431–4000)
Electrophoresis power supply (Fisher Scientific, cat. no. S65533Q)
Qubit Fluorometer (ThermoFisher Scientific, cat. no. Q33226)
Qubit assay tubes (ThermoFisher Scientific, cat. no. Q32856)
Agilent 4200 TapeStation (Agilent, cat. no. G2991AA)
High sensitivity D1000 ScreenTape (Agilent, cat. no. 5067–5584).
High sensitivity D1000 Reagents (Agilent, cat. no. 5067–5585).
Covaris LE220 Focused-ultrasonicator and chiller (Covaris, model no. LE220)
Covaris microTUBEs (Covaris, cat. no. 520052)
Covaris microTUBE rack (Covaris, cat. no. 500282)
DynaMag-2 magnetic rack (Life Technologies, cat. no. 12321D)
HiSeq 4000 System (Illumina)
Pippin Prep DNA Size Selection System (Sage Science, cat. no. PIP0001)
CFX96 Touch Real-Time PCR Detection System (BioRad, cat. no. 1855195)
6
Quantifying Extracellular DNA in Cultured Neutrophils
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Human neutrophils (5 × 105) were cultured in 24-well culture plates under the designated condition. The medium was centrifuged at 400×g for 5 min and the supernatant was obtained. The concentrations of total extracellular DNA in the cell culture supernatant were quantified by Qubit 3 fluorometer (ThermoFisher Scientific, MA, USA) and dsDNA HS assay kit (ThermoFisher Scientific, MA, USA). The reaction mixture for each sample consisted of 20 μL of the culture medium and 180 μL of working solution (dsDNA HS reagent:dsDNA HS buffer = 1:200) was prepared in Qubit Assay Tubes (ThermoFisher Scientific, MA, USA).
7
Qubit Fluorometric Analysis of gDNA
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To study the relation of homogenization method to Kit, samples were quanti ed by Qubit Fluorometer 3.0 (invitrogen Life Technologies, US). Qubit™ ds DNA HS standard 1 and Qubit™ ds DNA HS standard S2 were prepared by adding 189ul of buffer with 1ul of uorescent dye and 10ul of each standard provided with the Kit [11] (link). For sample preparation, 198ul of buffer was dispensed into tube (Qubit Assay Tubes, Thermo Fisher SCIENTIFIC) containing 1ul of dye. 1ul of extracted sample was added and the tubes were then incubated at room temperature (25 o C) for 1 minute and inserted in Qubit uorometer for observation. Thus, the quality of the isolated gDNA was evaluated by preparing 1.5% agarose gel electrophoresis.
8
Quantitative Bead-Based Fluorometric Assay
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Roughly 3,000 DART-seq beads were added to a mixture containing 18 uL of 5M NaCl, 2 μL of 1M Tris HCl pH 8.0, 1 μL of SDS, 78 μl of water, and 1 μL of 100 μM Cy5 fluorescently labeled oligo (see Supplementary Table ). The beads were incubated for 45 minutes at 46 ˚C in an Eppendorf ThermoMixer C (15”, at 1800 RPM). Following incubation, the beads were pooled and washed with 250 μL TE-SDS, followed by 250 μL TE-TW. The beads were suspended in 200 μl of DNAse/RNAse free water and transferred to a Qubit assay tube (ThermoFisher Scientific, Q32856). Qubit 3.0 Fluorometer was set to “Fluorometer” mode under the “635 nm” emission setting. The tube was vortexed briefly and placed in the fluorometer for immediate readout. Two additional vortexing and measurement steps were performed.
9
Quantitative Bead-Based Fluorometric Assay
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