Cell monolayers were infected with the viruses listed in the related figure legends at an MOI of 5 and incubated at 37°C for 12 h (VACV) or 24 or 48 h (SARS-CoV-2) after infection. The total RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA concentrations were determined using the Qubit 4 system (Thermo Fisher) and diluted to a final concentration of 5 ng/μL. Ten nanograms of the RNA samples was analyzed by RNA electrophoresis using the high-sensitivity RNA ScreenTape system (Agilent) according to the manufacturer’s instructions, and rRNA was visualized using the 2200 TapeStation system (Agilent).
Qubit 4 system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Qubit 4 system is a fluorometer designed for accurate and sensitive quantification of DNA, RNA, and protein samples. It measures the fluorescence of dye-labeled molecules to determine their concentration in a sample. The Qubit 4 system provides reliable results with small sample volumes and supports a wide range of applications in molecular biology and biochemistry research.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
2200 tapestation system, by Agilent Technologies (2 mentions)
Rna screentape system, by Agilent Technologies (2 mentions)
Nebnext companion module for technologies ligation sequencing, by Oxford Nanopore (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Candida parapsilosis, by American Type Culture Collection (1 mentions)
Acinetobacter baumannii, by American Type Culture Collection (1 mentions)
E faecalis, by American Type Culture Collection (1 mentions)
Candida tropicalis, by American Type Culture Collection (1 mentions)
Candida albicans, by American Type Culture Collection (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Optima xl 100k, by Beckman Coulter (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
5 protocols using qubit 4 system
1
Quantitative RNA Analysis of Virus-Infected Cells
2
Quantitative RNA Analysis of Virus-Infected Cells
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Cell monolayers were infected with the viruses listed in the related figure legends at an MOI of 5 and incubated at 37°C for 12 h (VACV) or 24 or 48 h (SARS-CoV-2) after infection. The total RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA concentrations were determined using the Qubit 4 system (Thermo Fisher) and diluted to a final concentration of 5 ng/μL. Ten nanograms of the RNA samples was analyzed by RNA electrophoresis using the high-sensitivity RNA ScreenTape system (Agilent) according to the manufacturer’s instructions, and rRNA was visualized using the 2200 TapeStation system (Agilent).
3
Barcoded Nanopore Library Preparation
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The amplicons were purified with CleanNGS magnetic beads (Labgene Scientific, Switzerland) and quantified with the Qubit 4 system (Thermo Fisher Scientific, USA). Twenty-four ng (2 pmol) of purified amplicon DNA were used for the barcoding procedure, using barcodes from the PCR Barcoding Expansion 1–96 kit (ONT, UK), and the amplification was carried out with the 2 × KAPA HiFi Hot Start Ready Mix kit (Roche, Switzerland). The cycling conditions were: first denaturation of 3 min at 95 °C, followed by 13 cycles of denaturation of 20 s at 95 °C, annealing of 15 s at 62 °C, elongation of 30 s at 72 °C, and a last elongation of 5 min at 72 °C. The barcoded DNA samples were subjected to a second step of magnetic beads purification and quantification, using the same methods as above, and combined in equal ratios to generate a pool.
One µg of pooled barcoded libraries was finalised using the Ligation Sequencing kit (ONT, UK) and the NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England Biolabs, USA). After a last step of magnetic beads purification and quantification, the libraries were loaded onto a R9.4 flow cell (ONT, UK) using the Flow Cell Priming Kit (ONT, UK).
One µg of pooled barcoded libraries was finalised using the Ligation Sequencing kit (ONT, UK) and the NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England Biolabs, USA). After a last step of magnetic beads purification and quantification, the libraries were loaded onto a R9.4 flow cell (ONT, UK) using the Flow Cell Priming Kit (ONT, UK).
4
RPA-LFS Detection of Pathogenic Bacteria
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In this study, E. faecalis, Acinetobacter baumannii, Candida parapsilosis, Candida tropicalis and Candida albicans were purchased from the American Type Culture Collection (Manassas, VA, USA). In addition, isolates of E. faecalis strains, isolates of other Enterococcus species, and isolates of other infectious pathogens were provided by The Second People’s Hospital of Lianyungang (Lianyungang, China). Sputum-isolated strains and clinical samples of sputum were collected from patients; 16S rRNA sequencing was performed to confirm the strains as described previously (Hiergeist et al., 2015 (link)). Information relating to the strains is given in Table 1
Clinical samples of sputum were treated at 100°C for 10 min. Furthermore, 1 µL of each samples was used as a template for the detection of RPA-LFS and qPCR methods.
Clinical samples of sputum were treated at 100°C for 10 min. Furthermore, 1 µL of each samples was used as a template for the detection of RPA-LFS and qPCR methods.
5
Isolation and characterization of small extracellular vesicles
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Passage 4 AD-MSC were cultured without serum in advanced Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with GlutaMAX™ (100 ×) (Thermo Fisher Scientific) for 48 h. The supernatant was collected and centrifuged at 2000 × g for 10 min to remove cell debris and filtered using a 0.22-μm filter (Stericup™ Quick Release Durapore™, Merck Millipore, Burlington, MA, USA). The filtered supernatant was ultracentrifuged at 210,000 × g for 70 min at 4 °C using an Optima XL-100 K (Beckman Coulter, Inc., Brea, CA, USA) with a swing rotor SW41Ti (Beckman Coulter, Inc.). The residual fraction was washed with phosphate-buffered saline (PBS; pH 7.4) and collected by ultracentrifugation (UC) at 210,000 × g. PBS was added to the final sEV-enriched fraction. Proteins were quantified using a Qubit4 system (Thermo Fisher Scientific).
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