DNA from environmental samples was extracted using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany) according to the manufacturer’s protocol. The concentration of the extracts was measured using the Qubit dsDNA HS kit (Thermo Fisher Scientific, USA, #Q33231) with a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA), while DNA purity was assessed with a NanoDrop One Spectrophotometer (Thermo Fisher, USA). Sample DNA fragment sizes were inspected using an Agilent 2200 Tapestation system with Genomic DNA ScreenTapes (Agilent Technologies, USA, #5067-5365). DNA samples were also size-selected (with the exception of BRK sample due to limited DNA amounts) using the Circulomics SRE XS kit (Circulomics, USA), following the manufacturer’s instructions to deplete DNA fragments below 10 kb.
Genomic dna screentape
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Genomic DNA ScreenTape is a lab equipment product from Agilent Technologies designed for the analysis of genomic DNA samples. It provides a quick and efficient method for assessing the size, concentration, and integrity of genomic DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation 2200, by Agilent Technologies (19 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (15 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (10 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (9 mentions)
Tapestation 4200, by Agilent Technologies (9 mentions)
Fastdna spin kit for soil, by MP Biomedicals (8 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (8 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (7 mentions)
Genomic tip 20 g, by Qiagen (7 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Genomic dna reagents, by Agilent Technologies (5 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Qubit dsdna hs br assay kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Buffer g2, by Qiagen (4 mentions)
Biomasher 2, by Funakoshi (4 mentions)
Buffer qf, by Qiagen (4 mentions)
High sensitivity d5000 screentape, by Agilent Technologies (4 mentions)
Dneasy blood and tissue kit, by Qiagen (4 mentions)
Tapestation system, by Agilent Technologies (4 mentions)
69 protocols using genomic dna screentape
1
Soil DNA Extraction and Purification
2
Soil DNA Extraction Protocol
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All samples were gently homogenized by stirring to obtain a representative subsample of approximately 0.5 g. Total genomic DNA was extracted using the FastDNA Spin kit for soil (MP Biomedicals) following the manufacturer's instructions, with hot phenol pre-treatment as described elsewhere (Albertsen et al., 2013 (link)). The quality of the DNA extracts was assessed using a TapeStation 2200 and Genomic DNA ScreenTapes (Agilent), and their concentration was estimated using a Qubit 3.0 fluorometer and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
3
DNA Extraction and Sequencing Workflow
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At the end of phase I and phase II, samples were collected for DNA extraction using the standard protocol for FAST DNA Spin kit for Soil (MP Biomedicals, United States) with the subsequent modifications; 500 μL of sample, 480 μL of sodium phosphate buffer, and 120 μL MT buffer were added to a Lysing Matrix E tube. Bead beating was done at a speed of 6 m/s for 4 x at 40 s each (Albertsen et al., 2015 (link)). Gel electrophoresis using Tapestation 2200 and genomic DNA screentapes (Agilent, United States) were performed to examine product size and purity of a subset of DNA extracts. DNA concertation was estimated using Qubit dsDNA HS/BR Assay kit (Thermo Fisher Scientific, United States).
The bacterial and archaeal 16S rRNA gene region V4 sequencing libraries were prepared by a custom protocol based on an Illumina protocol and the libraries were paired-end sequenced (2 bp × 300 bp) on a MiSeqTM (Illumina, United States). Details of library preparation are provided in the Supporting Information.
The shotgun metagenome sequencing library preparation was conducted using a TruSeq® Nano DNA LT Library Prep Kit according to manufacturer instructions (Illumina, United States). Libraries were quantified and normalized to 10 nM. The libraries were pooled and sequenced on HiSeqTM 4000 (Illumina, United States) (2 bp × 150 bp) paired-end platform at KAUST Bioscience Core Laboratory.
The bacterial and archaeal 16S rRNA gene region V4 sequencing libraries were prepared by a custom protocol based on an Illumina protocol and the libraries were paired-end sequenced (2 bp × 300 bp) on a MiSeqTM (Illumina, United States). Details of library preparation are provided in the Supporting Information.
The shotgun metagenome sequencing library preparation was conducted using a TruSeq® Nano DNA LT Library Prep Kit according to manufacturer instructions (Illumina, United States). Libraries were quantified and normalized to 10 nM. The libraries were pooled and sequenced on HiSeqTM 4000 (Illumina, United States) (2 bp × 150 bp) paired-end platform at KAUST Bioscience Core Laboratory.
4
Soil DNA Extraction with FastDNA Spin Kit
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DNA extraction was performed using the standard protocol for FastDNA Spin kit for Soil (MP Biomedicals) with the following exceptions. 500 L of sample, 480 L Sodium Phosphate Buffer and 120 L MT Buffer were added to a Lysing Matrix E tube. Bead beating was performed at 6 m/s for 4 × 40 s. Gel electrophoresis using Tapestation 2200 and Genomic DNA screentapes (Agilent) was used to validate product size and purity of a subset of DNA extracts. DNA concentration was measured using Qubit dsDNA HS/BR Assay kit (Thermo Scientific).
5
Comprehensive DNA Quality Assessment
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DNA concentrations were determined fluorometrically using Qubit® Fluorometric Quantitation dsDNA assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) on a Clariostar microplate reader (BMG Labtech, Ortenberg, Germany). All samples were diluted to 5 ng/µl with PCR-grade water. Purity was assessed by measuring the absorbance at 260 and 280 nm wavelengths using Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Maryland, USA). The optimal values of the absorbance ratios were expected to be in the range of 1.7–2.0 (A260/A280) and 1.8–2.2 (A260/A230). DNA integrity (DIN) was estimated automatically by running 1 µl of the gDNA samples on a 4200 TapeStation System, (G2991AA, Agilent Technologies; Santa Clara, California, United States) by using Genomic DNA ScreenTapes (5067–5365) and Agilent Genomic DNA reagents. The purified DNA samples were stored at −20 °C. For data analysis Analysis of Variance (ANOVA) test with Bonferroni corrections were conducted to compare the yield and purity of the nucleic acids such as the representative DIN values associated with the different technical variables.
6
Genomic DNA Extraction from Whole Blood
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Genomic DNA was obtained using 200 μl of the remaining whole-blood solid mass fraction in the Vacutainer tubes. Extractions were performed using the Qiagen DNA Mini Blood kit vacuum protocol (Qiagen, Hilden, Germany). All gDNA extractions were evaluated for fragment lengths and DNA integrity number using the Agilent Tapestation 4200 combined with Genomic DNA Screentapes (Agilent, Santa Clara, CA, USA) and stored at −80°C until sequencing.
7
MNase Digestion and Chromatin Analysis
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70–100 control or HisC mutant embryos were collected and lysed in 50 µl micrococcal nuclease (MNase) digestion buffer (15 mM Tris, pH 7.5; 15 mM NaCl; 60 mM KCl; 0.34 M sucrose; 0.5 mM spermidine; 0.15 mM spermine; 1 mM PMSF; 0.5 mM DTT; 0.1% β-mercaptoethanol, protease inhibitor [Roche]). 200 µl MNase digestion buffer and 1 mM of CaCl2 was added. The suspension was divided in equal aliquots. 500 gel units of MNase (NEB, Hitchin, UK) were added and the samples were incubated at 32°C in a heat block. The reaction was stopped by adding 5 µl of both 0.5 M EDTA and 0.5 M EGTA. 2.5 µl of 10% SDS and 1 µl Proteinase K (10 mg/ml) was added and incubated at 50°C over night. The DNA was purified using Ampure XP beads (Beckman Coulter, Brea, CA). The samples were analysed using an Agilent 2200 Tapestation system with High Sensitivity D1000 screen tapes (Agilent Technologies, Wokingham, UK). Genomic DNA screen tapes (Agilent Technologies) were used to determine the input (time point 0′) concentration. The Tapestation analysis software was used for quantification of following fractions: 60–115 bp (<115 bp), 115–270 bp (mononucleosomes), 270–455 bp (dinucleosomes), 455–650 bp (trinucleosomes).
8
Optimizing Soil DNA Extraction Protocols
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The standard protocols for DNA extraction with FastDNA Spin kit for soil (MP Biomedicals, USA) and PowerLyzer PowerSoil DNA isolation kit (MoBio, USA) were used with the following exceptions. Samples were thawed at room temperature, and varying amounts of biomass (0.9–2.2 mg TS) were spun down (5 min at >10,000 x g) and re-suspended in the respective lysis or bead solutions before extraction. Bead beating was performed on a FastPrep FP120 (MP Biomedicals, USA) at different intensity settings (20–400 s at 4–6 m/s), following general operation recommendations. All extraction conditions were tested in triplicate.
Purity of the extracted DNA was evaluated spectrophotometrically with Nanodrop1000 using A260/230nm and A260/280nm (Thermo Fisher Scientific, USA). The quality of the extracted DNA was evaluated with agarose gel electrophoresis, using the Tapestation 2200 and Genomic DNA screentapes (Agilent, USA). Finally, the concentration was measured fluorometrically with Quant-iT HS DNA Assay (Thermo Fisher Scientific, USA) on an Infinite M1000 PRO (Tecan, Switzerland).
Purity of the extracted DNA was evaluated spectrophotometrically with Nanodrop1000 using A260/230nm and A260/280nm (Thermo Fisher Scientific, USA). The quality of the extracted DNA was evaluated with agarose gel electrophoresis, using the Tapestation 2200 and Genomic DNA screentapes (Agilent, USA). Finally, the concentration was measured fluorometrically with Quant-iT HS DNA Assay (Thermo Fisher Scientific, USA) on an Infinite M1000 PRO (Tecan, Switzerland).
9
Extracting Genomic DNA from Fungal Isolates
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Isolates were cultured on PDA and harvested and ground using liquid N2. Genomic DNA was extracted using a cetyltrimethyl ammonium bromide (CTAB) protocol [47 (link),48 (link)]. DNA samples were assessed by running on a 1% agarose gel for the presence of DNA shearing and RNA, while DNA integrity was evaluated using TapeStation and Genomic DNA ScreenTapes (Agilent, Santa Clara, CA, USA) and impurities were evaluated using DropSense 16 (Trinean, Pleasantan, CA, USA). gDNA was quantified using a Qubit® 2.0 Fluorometer (Invitrogen by Life Technologies, Carlsbad, CA, USA) before submission for in-house sequencing (Molecular Technologies Laboratory, Ottawa Research & Development Centre, Agriculture and Agri-Food Canada) where DNA libraries were prepared and loaded onto a FLO-MIN106 flow cell and run with a MinION (Oxford Nanopore Technologies, Oxford, UK) for 48 h.
10
Gut Microbiome Metaproteomics Reference Database
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To generate a project-specific reference database for metaproteomics, whole genome sequencing of the gut microbiome was performed. DNA was extracted from 4 cecal feces samples of the 4 experimental groups (WT vehicle and dapagliflozin, diabetic vehicle and dapagliflozin). DNA extraction of samples was done using the FastDNA Spin kit for Soil (MP Biomedicals). Biomass was homogenized in 1000 μL sodium phosphate buffer. Then, 500 μL sample, 480 μL sodium phosphate buffer, and 120 μL MT buffer were added to a Lysing Matrix E tube. Bead beating was performed at 6 m/s for 4 cycles of 40 seconds each. Gel electrophoresis using Tapestation 2200 and Genomic DNA Screentapes (Agilent) was used to validate product size and purity of a subset of DNA extracts. DNA concentration was measured using the Qubit dsDNA HS/BR Assay kit (Thermo Fisher Scientific).
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