One milliliter of rumen sample was centrifuged at 13,000 rpm for 15 min, and the remained pellets were used for DNA extraction using i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc.) according to the manufacturer's instructions. Then, DNA quantity and quality were checked by agarose gel and Nanodrop spectrophotometer. Archaeal 16S rDNA gene was amplified using primers Ar915aF (5-AGG AAT TGG CGG GGG AGC AC-3) and Ar1386R (5-GCG GTG TGT GCA AGG AGC -3) (Rabee et al. 2022) (link). The PCR conditions were as follows: 95 °C for 5 min; 30 cycles 95 °C for 20 s, 55 °C for 15 s, 72 °C for 5 min, and 72 °C for 10 min. The PCR amplicons were purified and sequenced using Illumina MiSeq sequencing.
I genomic stool dna extraction mini kit
Manufactured by iNtRON Biotechnology
The I-genomic Stool DNA Extraction Mini Kit is a laboratory product designed to extract DNA from stool samples. The kit provides the necessary reagents and protocols to efficiently isolate and purify high-quality DNA from a small amount of stool material for further analysis.
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5 protocols using i genomic stool dna extraction mini kit
1
Rumen Microbiome DNA Extraction and Sequencing
2
Rumen Bacterial 16S rRNA Profiling
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One milliliter of every rumen sample was centrifuged at 13,000 rpm, and the remained precipitate was used for DNA extraction by i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc.) according to the manufacturer’s instructions. DNA was eluted in 50µL elution buffer, and DNA quality and quantity were checked by agarose gel electrophoresis and Nanodrop spectrophotometer, respectively. The V4 region of the bacterial 16S rDNA gene was amplified using 515F and 926R primers38 (link). PCR amplification was conducted under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s; and 72 °C for 10 min. PCR products purification, preparation for sequencing using Illumina MiSeq system were conducted according to the protocol described by Comeau et al.39 (link) in Integrated Microbiome Resource (IMR, Dalhousie University, Halifax, NS, Canada). Briefly, PCR-amplicons were cleaned up and normalized using the high-throughput Invitrogen SequalPrep 96-well plate kit. Then, the samples were finally pooled to make one library for the sequencing.
3
Rumen Microbiome DNA Extraction and Sequencing
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DNA extraction from rumen samples was conducted using i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc.) as described by Rabee et al. [9] (link) and DNA was eluted in 50 µL buffer. The quality and quantity of extracted DNA were checked using gel electrophoresis and Nanodrop spectrophotometer. Subsequently, the archaeal 16S rDNA gene was amplified using primers Ar915aF (5-AGGAATTGGCGGGGGAGCAC-3) and Ar1386R (5-GCGGTGTGTGCAAGGAGC-3) [5] (link). The PCR amplification was carried out under the following conditions: 95 °C for 5 min; 30 cycles 95 °C for 20 s, 55 °C for 15 s, 72 °C for 5 min, and 72 °C for 10 min. The PCR-amplicons were purified and prepared for Illumina MiSeq sequencing according to the protocol of Comeau et al. [21] (link) in Integrated Microbiome Resource (Dalhousie University, Canada).
4
Rumen Microbiome DNA Extraction and 16S Sequencing
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One milliliter of rumen sample was centrifuged at 14,000× g. The remaining pellets were used for DNA extraction by i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc., Korea) according to the manufacturer’s instructions. DNA was eluted in 50 μL elution buffer, and DNA quantity and quality were checked by agarose gel electrophoresis and nanodrop spectrophotometer (Thermo Fisher Scientific, Madison, Wisconsin, USA). The V4 region of the bacterial 16S ribosomal DNA gene was amplified using primers 515F and 926R [33 (link)]. PCR amplification was conducted under the following conditions: 94°C for 3 min; 35 cycles of 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s; and 72°C for 10 min. PCR products purification and preparation for sequencing using Illumina MiSeq system were conducted according to the protocol described by Comeau et al. [34 ] in Integrated Microbiome Resource (Dalhousie University, Canada).
5
Rumen Microbiome DNA Extraction and 16S rRNA Sequencing
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One milliliter of rumen fluid was centrifuged at 13,000 rpm and the precipitated pellets were used for DNA extraction by i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc., Seongnam-si, South Korea) according to the manufacturer’s instructions. DNA was then eluted in 50 µL elution buffer and DNA quality and quantity were verified using agar gel electrophoresis and Nanodrop spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). DNA Amplicon libraries targeting the V4–V5 region of the 16S rRNA bacterial 16S ribosomal DNA gene were generated by PCR amplification using primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 926R (5′-CCGYCAATTYMTTTRAGTTT-3′) (Walters et al., 2015 (link)). PCR amplification was conducted under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s; and 72 °C for 10 min. PCR products’ purification, preparation for sequencing using Illumina MiSeq system were conducted according to the protocol described by Comeau, Douglas & Langille (2017) (link) in Integrated Microbiome Resource (Dalhousie University, Canada).
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