Crew members isolated a stool sample using a paper toilet accessory (DNA Genotek, OM-AC1). Stool was transferred into and OMNIgene•GUT tube (DNAgenotek, OMR-200) and an OMNImet•GUT tube (DNA Genotek, ME-200). Tubes were placed at −80°C for long-term storage. For nucleic acid extraction, 200uL of each tube was allocated for DNA extraction with the QIAGEN PowerFecal Pro kit and 200uL was allocated to RNA extraction with the QIAGEN PowerViral kit. The remaining sample was split into 500uL aliquots and re-stored at −80°C.
Powerfecal pro kit
Manufactured by Qiagen
Sourced in Germany
The PowerFecal Pro kit is a laboratory equipment designed for the extraction and purification of nucleic acids from a variety of fecal samples. It is a tool used in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Onestep pcr inhibitor removal kit, by Zymo Research (2 mentions)
Quant it dsdna high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Miseq sequencer, by Illumina (2 mentions)
Miseq reagent kit v2, by Illumina (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Cfx96 amplifier, by Bio-Rad (2 mentions)
Om ac1, by DNA Genotek (1 mentions)
Qiasymphony automated extractor, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Quant it br dsdna reagent kits, by Thermo Fisher Scientific (1 mentions)
7 protocols using powerfecal pro kit
1
Gut Microbiome Preservation and Extraction
2
Viral Concentration and Nucleic Acid Extraction
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Samples were homogenised, then 11 mL were centrifugated at 200,000 x g for 1 hour at 4°C using a XPN80 (Coulter Beckman, Fullerton, United States (US)) ultracentrifuge equipped with a swing rotor (SW41Ti). Viral pellets were resuspended in 200 μL of PBS 1X buffer. The viral concentrate was lysed and extracted using PowerFecal Pro kit (QIAGEN, Hilden, Germany) on a QIAsymphony automated extractor (QIAGEN, Hinden, Germany ), according to a modified manufacturer’s protocol using a larger volume of samples. Extracted nucleic acids were filtered through OneStep PCR inhibitor removal kit (Zymoresearch, Irvine, US), according to the manufacturer’s instructions.
3
Optimized DNA Extraction from Tissue
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All sample tissue DNA was extracted using PowerFecal Pro kits (Qiagen) according to manufacturer’s protocol, with the exception that samples were homogenized using a TissueLyser II (Qiagen) for 10 min at 30/sec, in lieu of a vortex adapter as described by PowerFecal Pro kit instructions. DNA yields were quantified by fluorometry via the quant-iT BR dsDNA reagent kits (Invitrogen) and normalized to a consistent concentration and volume prior to submission for downstream processing.
4
16S rRNA Amplicon Sequencing of Gut Microbiome
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The samples were processed in two batches using the following protocol. DNA was extracted from stool and biopsy samples using the Qiagen Power Fecal PRO kit according to the manufacturer’s instructions. Amplification of V4 of the 16S rRNA gene was conducted using the following primers: modification of 515F (5′-GTGBCAGCMGCCGCGGTAA-3′) [21 (link)] and Pro-mod-805R (5′-GACTACNVGGGTMTCTAATCC-3′) [22 (link)]. The second amplification round was performed using standard Illumina indices with adapters. Both PCR rounds were done using the PCR buffer (Evrogen, Russia) and a Bio-Rad CFX-96 amplifier. PCR products were purified with the DNA Cleanup Mini kit (Evrogen, Russia). DNA concentration was measured with a Qubit fluorometer (Invitrogen, USA) using a Quant-iT dsDNA High-Sensitivity Assay Kit. Purified amplicons were mixed equimolarly according to the obtained concentrations. Further library preparation and sequencing were conducted using MiSeq Reagent Kit v2 (500 cycles) and MiSeq sequencer (Illumina, USA) according to the manufacturer’s recommendations. Primary processing (barcodes extraction) was done as described previously [23 (link)]. The post-quality trimmed read pairs were merged with SeqPrep; the resulting average read length was 252 bp.
5
Gut Microbiome Profiling by 16S rRNA Sequencing
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Stool samples were collected using DNA-stabilizing KnomX gut microbiome collection kits (KnomX, Moscow, Russia) stored at −30 °C. DNA extraction from the stool samples was carried out using the Qiagen Power Fecal PRO kit according to the manufacturer’s instructions. Amplification of the V4 region of the 16S rRNA gene was performed using modified 515F and 805R primers. The second round of amplification was performed using standard Illumina indexes with adapters. Both rounds of PCR were performed using the Eurogen PCR buffer and the Bio-Rad CFX-96 amplifier. PCR products were purified using the Cleanup Mini kit for DNA extraction (Evrogen, Moscow, Russia). The DNA concentration was determined using a Qubit fluorometer (Invitrogen, Waltham, MA, USA) and the Quant-iT dsDNA High-Sensitivity Assay Kit. Purified amplicons were mixed equimolarly according to the obtained concentrations. Further preparation of the samples for sequencing and sequencing of the pooled library was performed using the MiSeq Reagent Kit v2 (500 cycles) and the MiSeq sequencer (Illumina, San-Diego, CA, USA) according to the manufacturer’s recommendations. Primary processing (barcode extraction) was performed as previously described [29 (link)]. After quality trimming, reads were merged using the SeqPrep package; the total length of the merged reads was 252 bp.
6
Viral Concentrate Nucleic Acid Extraction
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The whole viral concentrate (~200µL) was lysed and extracted using Qiasymphony PowerFecal Pro kit on a QIAsymphony automated extractor (QIAGEN) according to a modified manufacturer’s protocol. Extracted nucleic acids were filtered through OneStep PCR inhibitor removal kit (Zymoresearch) according the manufacturer’s instructions for handling larger volumes.
7
Metagenomic Analysis of Fermented Foods
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In this study, 24 samples from various environments were analyzed (Tab. 1).
Fermented food samples were purchased from local markets or home-fermenting individuals in three countries in South-East Asia: Vietnam, Indonesia, and Malaysia.
Coconut milk yogurt, Greek yogurt and kefir, all labelled to contain live cultures were obtained from an organic store in Singapore. Aliquots of a total volume of approximately 20 ml were freeze-dried using a VIRTIS lyophilizer (SP Industries, Gardiner, NY, USA) and subsequently homogenized using a coffee grinder (CoffeeMill, Severin, Germany), which was thoroughly sterilized in-between samples.
Nucleic acids were extracted from 30 mg of food-associated sample as described above for lactobacilli isolates by using the combined bead-beating and Maxwell DNA extraction protocol.
To test applicability of the method to host-associated samples, a fresh fecal sample each was collected from a free-ranging wild boar (Singapore, NParks research permit number 18-075) and a pet guinea pig (Singapore). Nucleic acids were extracted with the Qiagen Power Fecal Pro kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. In addition, DNA from the jejunum content of a laboratory mouse fed on a standard chow diet was extracted according to Rius et al.
(40, kindly provided by Henning Seedorf, Temasek Life Sciences Laboratory, Singapore).
Fermented food samples were purchased from local markets or home-fermenting individuals in three countries in South-East Asia: Vietnam, Indonesia, and Malaysia.
Coconut milk yogurt, Greek yogurt and kefir, all labelled to contain live cultures were obtained from an organic store in Singapore. Aliquots of a total volume of approximately 20 ml were freeze-dried using a VIRTIS lyophilizer (SP Industries, Gardiner, NY, USA) and subsequently homogenized using a coffee grinder (CoffeeMill, Severin, Germany), which was thoroughly sterilized in-between samples.
Nucleic acids were extracted from 30 mg of food-associated sample as described above for lactobacilli isolates by using the combined bead-beating and Maxwell DNA extraction protocol.
To test applicability of the method to host-associated samples, a fresh fecal sample each was collected from a free-ranging wild boar (Singapore, NParks research permit number 18-075) and a pet guinea pig (Singapore). Nucleic acids were extracted with the Qiagen Power Fecal Pro kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. In addition, DNA from the jejunum content of a laboratory mouse fed on a standard chow diet was extracted according to Rius et al.
(40, kindly provided by Henning Seedorf, Temasek Life Sciences Laboratory, Singapore).
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