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7 protocols using glass beads

1

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).
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2

DNA Extraction from Fecal Samples

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Fecal specimens were stored at −80°C until use. The DNA from the fecal samples was recovered using the QIAamp DNA Stool kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions with some modification (10 (link)). Two hundred milligrams of the fecal specimens was weighed in a 2 ml microcentrifuge tube containing 0.3 g glass beads, and 180 µl of ATL lysis buffer solution was added (Qiagen). The suspension was mixed using a vortex mixer followed by bead beating three times for 30 s at a setting of 4,200 rpm using a bead beater (Yasui Kikai, Tokyo, Japan). In addition, 20 µl of proteinase K was added to the mixture. After heating at 56°C for 30 min, the bacterial cells in the samples were treated by bead beating in the same manner. After destruction of bacterial cells, total DNA was collected using a Qiagen column and purified according to the manufacturer’s instructions. The DNA was extracted in 200 µl of AE buffer (Qiagen) with 400 µl of ethanol and 20 µl of 3 M sodium acetate and kept at −20°C for 14–16 h for ethanol precipitation. The precipitated DNA was collected by centrifugation for 20 min at 20,000 × g.
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3

TNFα-stimulated HUVEC chromatin analysis

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Approximately 5 × 106 HUVECs stimulated with TNFα for 0, 10 or 30 min were digested (3 min at 37°C) with 750 units of micrococcal nuclease (MNase; Sigma-Aldrich, Seelze, Germany). Mononucleosomal DNA was isolated following separation on 1.3% agarose gels using glass beads (Qiagen, Hilden, Germany), and average fragment lengths determined using a 2100 Bioanalyzer (Agilent). Libraries were generated using the NEBNext DNA Library Prep Master Mix Kit (New England Biolabs, Ipswich, USA) and paired-end (2 × 50-bp) sequenced on a HiSeq2000 platform (Illumina, Essex, UK) to comparable depths (that is, 181, 185 and 187 million reads for 0, 10 and 30 min samples, respectively). Obtained reads were processed using the toolkits FastQC [44 ] and FASTX [45 ], mapped to hg19 using Bowtie [46 (link)].
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4

Efficient Cell Lysis for Microbial Analysis

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After the host biomass depletion methods (Saponin, Cytolysis or MolYsis), the pellets containing bacterial or fungal cells were resuspended in 150 µl of PBS. The cell suspensions were transferred to 200 µl vials containing glass beads (Sigma-Aldrich, G1145). The cells were lysed by bead-beating, using approx. 50 μl of acid-washed 150-212 μm glass beads in a 200 μl tube, with a TissueLyser (Qiagen, 85220) with the following settings: frequency 1/25 s and 5 minutes. The cell lysates were frozen at -20°C until analysis.
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5

Gut Microbiome Profiling via Stool Sampling

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Stool samples from subjects before and after MNT were collected uniformly by trained study personnel. We distributed disposable sterile bowls for stool collection to the subjects beforehand and introduced points to note. Subjects picked up approximately 1 g of stool with a small spoon after a natural bowel movement. Fresh stool samples were collected from recruited subjects and transported to the laboratory in ice packs within 2 hours. All samples were then frozen immediately and stored at—80°C prior to analyses.
DNA was extracted from each fecal samples using improved protocol based on the manual of QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany). In detail, 1ml of InhibitEX Buffer and proper amount of glass beads (0.5mm diameter, Qiagen) was added to each 200mg of feces. The mixture was homogenized and beat with 60Hz for 1 min twice with a Homogeneous instrument (FASTPREP-24, Aosheng Biotech, China). Afterwards, the DNA purification was performed according to the manufacturer’s instructions.
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6

Fecal DNA Extraction from Mice

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The total fecal DNA of mice was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Briefly, 1 ml of EX inhibition buffer and an appropriate amount of glass beads (0.5 mm diameter, Qiagen) were added per 200 mg of feces; the mixture was then homogenized at 60 Hz twice per minute with a homogenizer (FASTPREP-24, Aosheng Biotech, China). The concentration of DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, United States).
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7

Fecal DNA Extraction Protocol

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Fresh fecal samples were collected from the participants on their first visit and before any pharmacotherapy treatment at the Centre for Reproductive Medicine. These samples were then transported to the laboratory with an ice pack within 2 hours. All samples were then snap frozen and stored at À80 C before analyses. We extracted DNA from each fecal sample using an improved protocol based on the QIAamp Fast DNA Stool Mini Kit instructions (Qiagen). In more detail, 1 mL of InhibitEX buffer and a sufficient number of glass beads (0.5 mm diameter; Qiagen) were added to each 200 mg of feces. The mixture was homogenized and beaten with 60 Hz for 1 minute, twice with a homogeneous instrument (FASTPREP-24; Aosheng Biotech). We subsequently performed DNA purification according to the manufacturer's instructions.
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