Approximately 5 mL of feces for each volunteer was collected using sterile fecal sampling tubes (SARSTEDT AG & Co. KG, Nümbrecht, Germany) to lower the risk of bias. All samples were stored at − 80 °C prior to isolating genomic DNA using the TIANamp Stool DNA Kit (Tiangen Angen Biotech (Beijing) Co., Ltd., Beijing, China) following the manufacturer’s instructions. DNA integrity was evaluated by agarose gel electrophoresis on a 1.2% agarose gel with 1 × TAE Buffer running at a constant voltage of 110 V. A Qubit R3.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) was used to assess the quality of the DNA and to measure DNA purity and concentration. DNA with an OD260/OD280 ratio between 1.8 and 2.0 was considered pure, and all of the DNA concentrations were higher than 2.5 ng/μL.
Qubit r3.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit R3.0 Fluorometer is a compact and precise instrument designed for quantifying nucleic acids, proteins, and other biomolecules. It utilizes fluorescence-based detection methods to provide accurate and reliable measurements. The Qubit R3.0 Fluorometer is a standalone device that can be used for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Nucleospin microbial dna kit, by Macherey-Nagel (1 mentions)
Novaseq 6000 technology, by Illumina (1 mentions)
Nanodrop 1000, by Ozyme (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Pacbio sequel sequencer, by Pacific Biosciences (1 mentions)
Truseq nano dna library prep kit, by Illumina (1 mentions)
4 protocols using qubit r3.0 fluorometer
1
Fecal DNA Extraction Protocol
2
Bacterial Genomic DNA Extraction and Sequencing
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Bacterial genomic DNA was extracted using the NucleoSpin® Microbial DNA Kit (Machery-Nagel, Hoerdt, France) according to the manufacturer’s instructions. DNA concentration and purity were respectively determined using the Qubit R 3.0 Fluorometer (Thermo Fisher Scientific, Illkirch, France) and the NanoDrop 1000 (Ozyme, Saint-Cyr-l’Ecole, France). Library preparation was performed using the Nextera XT DNA library preparation kit and 2 × 150 paired-end sequencing was performed using the NovaSeq 6000 Illumina technology (Illumina, San Diego, USA). Illumina adapter sequences were removed and reads were quality trimmed using trimmomatic version 0.39.23 (link)De novo assemblies were generated with Shovill version 1.0.0 (https://github.com/tseemann/shovill ), and the quality of assemblies was assessed using QUAST v4.5.1.24 (link)
3
Bacterial DNA Extraction from Neonatal Stool
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Meconium and fecal samples were collected from neonates and subsequently stored at − 80°C. Samples were subject to DNA extraction using QIAamp DNA Stool Mini Kit (Qiagen, West Sussex, UK) with a slight modification of the standard protocol: a supplementary incubation at 95 °C for five minutes of the stool sample with the lysis buffer was added to enhance the bacterial cell rupture [21 (link)]. The purity of the DNA was determined by measuring the ratio of the absorbance at 260 and 280 nm (Infinite R 200 PRO Nano Quant, Tecan, Mannedorf, Switzerland) and the concentration was evaluated by Qubit R 3.0 Fluorometer (Invitrogen, Life Technologies, CA, USA).
4
Weevil Genome DNA Sequencing
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High-molecular-weight genomic DNA was isolated from approximately 400 male and female weevils using a TreliefTM Animal Genomic DNA Kit (TsingKe, China), and the DNA quality and quantity was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and a Qubit(r) 3.0 Fluorometer (Invitrogen, USA). The extracted DNA was then used to construct Illumina libraries and PacBio RSII libraries. PE genomic libraries with an insertion length of 270 bp were constructed and sequenced on an Illumina HiSeq X Ten platform (Illumina, USA) according to the Illumina TruSeq Nano DNA Library Prep Kit; a total of 27.64 Gb of raw data of PE sequences (2 x 150 bp) forC. formicarius were obtained. For longread sequencing, single molecule real-time (SMRT) cell 20-kb DNA libraries were constructed on a PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA) according to the standard PacBio protocol; one movie of the SMRT cells was acquired at Biomarker Technologies Corporation (Beijing, China). The original data were subjected to strict quality control before assembly. For Illumina data, we used the Trimmomatic program v0.33 (Trimmomatic, RRID:SCR-011848) to remove adaptor sequences and trim low-quality reads (Bolger, Lohse, & Usadel, 2014) (link).
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