Fresh fecal pellets were collected for 16S rRNA analysis. Mice were removed from home cages and placed individually in clean cages without bedding and were provided with food and water. Pellets were collected at the same time of day within one hour for each mouse at every time point and stored at -20°C. Samples were collected at the beginning of the study, while mice consumed standard chow (T0). Immediately after this collection, mice were fed HF diet. Fecal samples were collected again after one, two, and three days consuming HF diet (T1, T2, T3, respectively) and at the end of the study, after consuming HF diet for five weeks (TT). Metagenomic DNA from mouse stool pellets was extracted using a modified Qiagen DNEasy protocol. A single stool pellet (10-60mg total weight) was mechanically lysed using Qiagen PowerBead garnet tubes with 1mL of Qiagen InhibitEX buffer. The lysate was then further processed and metagenomic DNA was extracted onboard a QiaCube HT instrument. The 16S rRNA V1-V3 region was amplified using primers 27F (5’-AGAGTTTGATCCTGGCTCAG ) and 534R (5’-ATTACCGCGGCTGCTGG ) that incorporated Illumina dual indices and sequencing adapters. The amplicons were pooled for sequencing using Illumina 2x300 base V3 reagents on a MiSeq instrument at The Jackson Laboratory for Genomic Medicine Genome Technologies core facility.
Inhibitex buffer
Manufactured by Qiagen
Sourced in Germany, United Kingdom, United States, Netherlands
InhibitEX buffer is a component of Qiagen's sample preparation kits. It is designed to inhibit PCR inhibitors present in the sample, allowing for more efficient nucleic acid extraction and purification.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp fast dna stool mini kit, by Qiagen (19 mentions)
Qiamp fast dna stool mini kit, by Qiagen (5 mentions)
Proteinase k, by Qiagen (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Glass beads, by Qiagen (3 mentions)
Qiaamp dna stool mini kit, by Qiagen (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Zirconia bead mix, by Benchmark Scientific (2 mentions)
Beadbug microcentrifuge homogenizer, by Benchmark Scientific (2 mentions)
Qubit fluorimetric quantitation, by Thermo Fisher Scientific (2 mentions)
Magna lyser, by Roche (2 mentions)
Sterile polypropylene microvials, by Biospec (2 mentions)
Glass bead, by Biospec (2 mentions)
Zirconia silica bead, by Biospec (2 mentions)
Mini beadbeater 16, by Biospec (2 mentions)
Nanodrop uv spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Bead ruptor elite bead mill homogenizer, by Omni International (2 mentions)
0.1 mm glass beads, by Biospec (2 mentions)
38 protocols using inhibitex buffer
1
Gut Microbiome Dynamics in Mice
2
Murine Intestinal Sampling Protocol
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Faecal samples were collected into sterile Eppendorf tubes and snap‐frozen on dry ice. Mice were then killed by CO2 inhalation. Small intestinal and distal colon snips were fixed in either Carnoy's fixative (60% ethanol absolute, 30% chloroform and 10% glacial acetic acid) to preserve the mucus or KP‐CryoCompound (VWR, Lutterworth, UK). The remaining colon was opened up and any remaining faecal matter was removed and gently washed away with phosphate‐buffered saline (PBS; Sigma, Poole, UK). The inner surface of the colon was scraped using a cell scraper and InhibitEX buffer (Qiagen, Manchester, UK) to remove mucus from the mucus lining, which was then snap‐frozen. Serum was incubated at 37° for 2 hr, before centrifugation at 7000 g for 10 min to collect the supernatant. The supernatant was stored at −80°.
3
Wastewater Nucleic Acid Extraction
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Once the final volume of concentrate was collected from each wastewater sample, the sample was pretreated with InhibitEX buffer (Qiagen Sciences, Maryland, MD) as indicated by the manufacturer. Then, QIAamp MinElute virus spin kit (Qiagen Sciences, Maryland, MD) was used to extract total nucleic acids from each wastewater sample as per the manufacturer’s instructions, which included the use of Qiagen Protease and carrier RNA (Qiagen Sciences, Maryland, MD). Samples were eluted in 75 µL of Buffer AVE (Qiagen Sciences, Maryland, MD), quantified, and stored at −80 °C for downstream processes. The nucleic acid concentration and purity were assessed using Qubit dsDNA high sensitivity and RNA assay kits in a Qubit 4 fluorometer (Invitrogen, Carlsbad, CA, USA). Qubit results can be found in Supplementary Materials (Table S1 ).
4
Fecal and Mucus DNA Extraction for Microbiome Analysis
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DNA extraction was performed using a Qiamp® Fast Stool DNA Mini Kit (Qiagen), using a modified version of the manufacturer's instructions. Faecal samples were incubated in Inhibitex buffer (Qiagen) and mechanically disaggregated, before incubation at 95° for 30 min. Mucus samples were centrifuged (13 000 g for 10 min) and the mucus pellets were incubated in Inhibitex buffer, mechanically disaggregated and incubated at 95° for 30 min. Three hundred microlitres of the resulting lysate was used for the subsequent steps, which were then performed according to the manufacturer's instructions. Optical density at 260 nm was recorded using a UV1101 spectrophotometer (Biochrom Ltd, Cambridge, UK) to measure DNA concentration. For the identification of different bacterial species, the 16S rRNA gene was amplified using the universal primer pairs P3_GC‐341F and P2_518 (Table 1 ), as described previously.7
5
Murine Fecal and Colonic Sampling
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Sample collection and processing was performed as described by Glymenaki et al.17 (link). In brief, samples were harvested from mice at two time points, 6 and 18 weeks of age. Stool samples were collected from mice in individual autoclaved cages into sterile tubes and snap frozen on dry ice. Mice were sacrificed via CO2 inhalation, the proximal colon was cut open and the colonic mucus scraped using cell scrapers and Inhibitex buffer (QIAGEN, Manchester, UK) and snap frozen until use. Genomic DNA was extracted using QIAamp Fast Stool Mini-Kits according to the manufacturer’s instructions (QIAGEN).
6
Comprehensive Fecal Microbiome Profiling
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DNA was extracted from each fecal sample using the modified protocol of the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Briefly, 1 ml InhibitEX buffer and glass beads (0.5-mm diameter, Qiagen) were added to a 200 mg fecal sample. The mixture was homogenized and mixed at 60 Hz for 1 min (twice) using a homogenizer. DNA was purified in accordance with the manufacturer's instructions.
The V3-V4 region of the bacterial 16S ribosomal RNA genes was amplified by PCR using barcoded primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′ (Wang and Qian, 2009 (link)). PCRs were performed in a volume of 30 μl containing 15 μl 2 × KAPA Library Amplification ReadyMix, 1 μl each primer (10 μM), 50 ng template DNA, and ddH2O (Pereira et al., 2017 (link)).
Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions, and quantified using Qubit®2.0 (Invitrogen, Waltham, MA USA). All quantified amplicons were pooled to equalize the concentrations for sequencing using the Illumina MiSeq PE250 (Illumina, Inc., San Diego, CA, USA). The paired-end reads (~250 bp in length) were overlapped at their 3′ ends for concatenation into original longer tags using PANDAseq (https://github.com/neufeld/pandaseq , version 2.9).
The V3-V4 region of the bacterial 16S ribosomal RNA genes was amplified by PCR using barcoded primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′ (Wang and Qian, 2009 (link)). PCRs were performed in a volume of 30 μl containing 15 μl 2 × KAPA Library Amplification ReadyMix, 1 μl each primer (10 μM), 50 ng template DNA, and ddH2O (Pereira et al., 2017 (link)).
Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions, and quantified using Qubit®2.0 (Invitrogen, Waltham, MA USA). All quantified amplicons were pooled to equalize the concentrations for sequencing using the Illumina MiSeq PE250 (Illumina, Inc., San Diego, CA, USA). The paired-end reads (~250 bp in length) were overlapped at their 3′ ends for concatenation into original longer tags using PANDAseq (
7
Fungal and Bacterial Fecal Composition
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To determine the fungal and bacterial composition of the fecal samples, the DNA was isolated using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Venlo, The Netherlands). Fungi have a stronger cell wall containing chitin; therefore, a bead-beating step was introduced in addition to the protocol from the kit as adapted from [17 (link)]. In short, 500 µL of fecal sample was transferred to a Precellys tube (Bertin Corp, Rockville, MD, USA) which contained 0.5 mm glass beads. Then 1 mL InhibitEX buffer (Qiagen) was added, and the samples were treated in the Precellys 24 homogenizer (Bertin Corp) at 6000RPM for 3 × 30 s. The samples were cooled on ice in between sessions. Next, the samples were heated to 95 °C for 7 min, subsequently mixed by vortexing, and the sample was pelleted by centrifugation for 1 min at 14,000× g. Thereafter, 30 µL proteinase K was added, and the steps were followed as described in the QIAamp Fast DNA Stool Mini Kit handbook. The final elution step was performed in the smaller volume of elution buffer (50 µL) to increase the DNA yield. After elution, the DNA concentration was determined using the Qubit 1X dsDNA high sensitivity (HS) assay and a Qubit 3.0 Fluorometer (Invitrogen, Waltham, MA, USA).
8
Optimized Stool DNA Extraction for Microbiome Analysis
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DNA was extracted using the QIAquick FAST DNA Stool kit (QIAGEN, Mississauga, Canada) with these modifications: ~250mg of frozen stool was suspended in 1.4mL of Inhibitex buffer (QIAGEN) supplemented with 50μg of lysozyme (Sigma) and 13.5U of lysostaphin (Sigma), then subjected to 5 minutes of bead-beating with 500mg of 0.1mm silica/zirconium beads (BioSpec Corp) on a Vortex Genie with a MoBio adapter. The supernatant was incubated at 70°C for 10 minutes with 100μg Proteinase K QIAGEN) and 400μg RNase A (Sigma), microfuged for 1 minute and transferred to a clean tube, after which the manufacturer’s protocol was resumed. DNA was eluted in 100μL of buffer. If the concentration as measured on a Nanodrop 2000 was <10μg/mL, the extraction was repeated. A 50μL portion of the extracted DNA was removed for qPCR analysis and the remainder was used for 16S rDNA amplicon generation.
9
Fecal Microbiome Profiling Protocol
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Participants were instructed to collect fecal samples in their households. Sterile stool collection kits, containers, and temperature-controlled packages were provided. A fresh stool was placed into a half of 50 ml sterile container and double-sealed in a zip-lock bag. The sample was stored in the freezer compartment of a home refrigerator, delivered to the laboratory within 24 hours, and stored at −80°C until use. Fecal samples were resuspended in InhibitEX Buffer (QIAGEN, Germany), incubated at 70°C for 5 min, and centrifuged at 20,000 × g for 1 min. Supernatant was collected for DNA isolation using QIAamp fast DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. DNA integrity and concentration were quantitated using the Qubit dsDNA HS assay (Life Technologies, USA) on a Qubit 4 Fluorometer (Life Technologies, USA). The V3–V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene was PCR-amplified using specific primers [20 (link)]. The PCR products were subjected to gel purification using the NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions.
10
Microbial DNA Extraction and Sequencing
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DNA was extracted from both the mouth and posterior intestine using QIAamp DNA stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The collected samples were transferred to a 5 ml tube that contained 1.4 mm zirconium oxide beads (Cayman chemical, Ann Arbor, MI, United States). Then, 2 ml of InhibitEX buffer (Qiagen) was added to the tube. The extracted DNA was eluted in 75 μl ATE buffer. Thereafter, the quality and quantity of the extracted DNA were checked with NanoDrop spectrophotometer ND-8000 (Thermo Fisher Scientific Inc., Waltham, MA, United States).
Prior to library preparation, the extracted DNA from each sample was treated with REPLI-G kit (Qiagen) to enrich the microbial DNA. Library preparation and sequencing were performed as described previously (Abdelhafiz et al., 2021a (link)).
Prior to library preparation, the extracted DNA from each sample was treated with REPLI-G kit (Qiagen) to enrich the microbial DNA. Library preparation and sequencing were performed as described previously (Abdelhafiz et al., 2021a (link)).
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