DNA extraction was performed with PowerSoil DNA Isolation Kit (Qiagen) following the manufacturer’s instructions and optimized with phenol‒chloroform. Briefly, sample batches (approximately 250 μL) were vortexed for 10 min in bead tubes with 60 μL of C1 solution, 100 μL chloroform (Sigma‒Aldrich), and 100 μL phenol (Sigma‒Aldrich). Next, 16S ribosomal amplicons were generated using the 341F (5’-CCTACGGGNGGCWGCAG-3’ ) and 805R (5’-ACTACHVGGGTATCTAATCC-3’ ) primers. These amplified V3-V4 regions had a fragment size of approximately 464 bp. Amplicon sequencing was performed using the MiSeq Illumina platform (2x300 bp). Microbial DNA enrichment was performed for shotgun sequencing using the NEBNext® Microbiome DNA Enrichment Kit (New England Biolabs, NEB) following the manufacturer’s instructions. For shotgun sequencing, the Illumina NextSeq system (2x150) was utilized. Both 16S amplicon and shotgun sequences were generated using the LABSERGEN Langebio Cinvestav Irapuato platform.
Phenol
Manufactured by Qiagen
Phenol is a chemical compound that is commonly used in molecular biology and biochemistry laboratories. It is a colorless crystalline solid with a distinctive odor. Phenol is primarily used for the extraction and purification of nucleic acids, such as DNA and RNA, from biological samples. It is an effective denaturant that helps to separate proteins from nucleic acids, allowing for the isolation and purification of the genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext microbiome dna enrichment kit, by New England Biolabs (1 mentions)
Phenol, by Merck Group (1 mentions)
Miseq platform, by Illumina (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Chloroform, by Merck Group (1 mentions)
Chloroform, by Qiagen (1 mentions)
Rnase a, by Qiagen (1 mentions)
Phenol, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
2 protocols using phenol
1
Optimized DNA Extraction and Microbiome Sequencing
2
Efficient DNA Extraction via Reverse Crosslinking
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Cell lysis and protease digestion were done at 55°C with constant mixing in 10 mM Tris, 10 mM EDTA, 0.5% SDS, and 500 µg/ml proteinase K (NEB) for 8–12 h (107 cells/ml). NaCl was added for a final 0.2 M concentration, and the lysate was incubated 20–24 h at 65°C with constant mixing to reverse formaldehyde cross-linking. DNA was extracted 2× using an equal volume of phenol (Thermo Fisher Scientific)/chloroform/isoamyl alcohol (25:24:1) and 1× with an equal volume of chloroform/isoamyl alcohol (24:1). Tubes were kept at 55°C for 1 h with the cap off to evaporate phenol residues, and then they were treated with 25 µg/ml RNaseA (QIAGEN) at 37°C for 1 h and extracted again. DNA was then precipitated with 1:10 volume of 3 M Na acetate, pH 5.2, and two volumes of 100% EtOH at −20°C for 20–120 min.
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