DNA was extracted from the Pakistani and Finnish participants’ fecal samples using the same procedure, Repeated Bead Beating (RBB) method,73 (link) with the following modifications for automated DNA purification: Approximately, 0.25 g of fecal samples and 340 µL and 145 µL of lysis buffer were used on the first and second rounds of bead beating, respectively. Then, 200 µL of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ −96 DNA Multi-Sample Kit (4413022, Thermo Fisher Scientific, USA) using the KingFisherTM Flex automated purification system (ThermoFisher Scientific, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). Library preparation and Illumina MiSeq sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene using primers 341 F/785 R were performed as previously described.74 (link) To characterize the gut fungal community, we performed ITS-1 sequencing for the Pakistani samples using a two-step PCR protocol described in detail elsewhere.75 (link) PCR-amplicons of the ITS-1 region were generated using ITS1F and ITS2 primers.75 (link),76 (link)
Ambion magmax 96 dna multi sample kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion Magmax™ −96 DNA Multi-Sample Kit is a lab equipment product designed for the isolation and purification of DNA from various sample types. It utilizes magnetic bead-based technology to enable high-throughput and automated DNA extraction.
Automatically generated - may contain errors
Lab products found in correlation
Quanti it pico green dsdna assay, by Thermo Fisher Scientific (2 mentions)
Kingfisher flex automated purification system, by Thermo Fisher Scientific (2 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Kingfisher flex, by Thermo Fisher Scientific (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
3 protocols using ambion magmax 96 dna multi sample kit
1
Metagenomic Profiling of Gut Microbiome
2
Collection and Extraction of Fecal and Serum Samples
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Fecal samples for the patients and controls were collected at home and immediately stored at -20°C. They were transported to a study center within 1 week. An uninterrupted frozen cold chain was ensured in the provision and handling of the fecal samples. Bacterial DNA was extracted from ca. 250 mg of fecal matter using the Repeated Bead Beating (RBB) method (13 (link)) with the following modifications for automated DNA purification: 340 μl and 145 μl of lysis buffer was added to first and second round of bead beating, respectively. 200 µl of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ -96 DNA Multi-Sample Kit (Thermo Fisher Scientific, USA) using the KingFisherTM Flex automated purification system (Thermo Fisher Scientific, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA).
Blood samples were collected in the morning between 7 and 10 am after an 8- to 12-hour fast before taking morning medications. Sera were isolated with a standard protocol and stored at -80°C until analyses.
Blood samples were collected in the morning between 7 and 10 am after an 8- to 12-hour fast before taking morning medications. Sera were isolated with a standard protocol and stored at -80°C until analyses.
3
Analyzing Gut Microbiome Using 16S rRNA Sequencing
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Bacterial DNA was extracted from ca. 50 mg of fecal samples and 30–50 mg of cecal content using the Repeated Bead Beating (RBB) method for automated DNA purification.92 (link) 200 µL of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ −96 DNA Multi-Sample Kit (4413022, Thermo Fisher Scientific, USA) using the KingFisher™ Flex automated purification system (Thermo Fisher Scientific, USA). DNA was quantified using Quant-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). Library preparation and Illumina MiSeq sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene were performed as previously described.93 (link)Sequences were processed using QIIME2 v.2020.11 pipeline.94 (link) Demultiplexed 250-bp paired-end sequences were denoised using DADA2 to obtain an amplicon sequence variant (ASV) table.95 (link) Singletons (ASV present <2 times) and ASVs present in less than 10% of the samples were discarded. Taxonomic classification was performed using a pre-trained naive Bayes classifier implemented in QIIME2 against the SILVA 132 reference database.96 (link) Taxa that could not be identified at genus-level are referred to as the highest taxonomic rank identified. Samples meeting quality criteria (107 out of 108) had a mean sequencing depth of 11952 reads.
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