We collected stool samples from 16-wk-old female Kcasp1Tg mice and wild type (WT) littermate mice. DNA was extracted using the MPure Bacterial DNA extraction kit (MP Biomedicals, Irvine, CA, USA). Sequencing was performed on the Illumina’s MiSeq platform (Illumina, San Diego, CA, USA) using a 2 × 300 bp run. Sequencing data were processed using the QIIME (Quantitative Insights Into Microbial Ecology) bioinformatics pipeline. Analysis of our next generation sequencing data revealed the microbiome to the genera level. Stool samples of Kcap1Tg mice were also analyzed using the GRIDion X5 system by Oxford Nanopore Technologies (Oxford Science Park, Oxford, UK), which enabled us to analyze our data to the species level. Data were analyzed using the USEARCH software and the RDP_16S_V16_sp database.
Mpure bacterial dna extraction kit
Manufactured by MP Biomedicals
Sourced in United States
The MPure Bacterial DNA Extraction Kit is a product designed for the efficient isolation and purification of bacterial DNA from various sample types. The kit utilizes a proprietary extraction method to obtain high-quality DNA suitable for downstream molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Lysis solution f, by Nippon Gene (3 mentions)
Synergy h1, by Agilent Technologies (3 mentions)
Vd 250r freeze dryer, by TAITEC (2 mentions)
Quantifluor dsdna system, by Promega (2 mentions)
Mpure 12 system, by MP Biomedicals (2 mentions)
Synergy lx, by Agilent Technologies (2 mentions)
Mpure 12 automated nucleic acid purification system, by MP Biomedicals (2 mentions)
Gridion x5 system, by Oxford Nanopore (1 mentions)
Miseq platform, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Vd 250r, by TAITEC (1 mentions)
8 protocols using mpure bacterial dna extraction kit
1
Analyzing Gut Microbiome in Kcasp1Tg Mice
2
Extraction and Purification of Bacterial DNA from Root Caries Samples
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The collected dentin shavings were freeze-dried (VD-250R Freeze Dryer, TAITEC, Saitama, Japan) and pulverized at 1500 rpm for 2 min (Shake Master Neo, Bio Medical Science, Tokyo, Japan). The powdered root caries samples were added to a lysis solution (Lysis Solution F, Nippon Gene, Tokyo, Japan) and left to stand for 10 min at 65 °C. The samples were centrifuged at 12,000× g for 1 min, and genomic DNA was extracted using an MPure Bacterial DNA Extraction Kit (MP Biomedical, Santa Ana, CA, USA).
3
DNA Extraction from Fecal Samples
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Fecal samples were frozen and dried using VD-250R Freeze Dryer (TAITEC Corp., Saitama, Japan) and were mechanically disrupted using Shake Master Neo (Bristol-Myers Squib. Co., Ltd., NY, USA). Total DNA was extracted using MPure Bacterial DNA Extraction Kit (MP Biomedicals Japan. Co., Ltd., Tokyo, Japan). The concentration of the extracted DNA was determined by using a Synergy H1 (Bio Tek, Instruments, Inc., Winooski, VT, USA) and QuantiFluor dsDNA System (Promega Corp., Madison, WI, USA), and samples were stored at −30°C until use.
4
Fecal DNA Extraction Protocol
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Faecal samples were collected using a guanidine thiocyanate solution (Faeces Collection kit; Techno Suruga Lab, Shizuoka, Japan). After vigorous mixing, the samples were stored at 4 °C for a maximum of 7 days until DNA extraction. After homogenisation with lysis solution F (Nippon Gene, Tokyo, Japan), the genomic DNA was heated at 65 °C for 10 min and purified from the supernatants using the MPure Bacterial DNA Extraction Kit (MP Biomedicals, Solon, OH, USA) with MPure-12 system (MP Biomedicals). The purified DNA was quantified using Synergy LX (BioTek Instruments, Winooski, VT, USA) and QuantiFluor system (Promega, Madison, WI, USA).
5
Freeze Drying and DNA Extraction
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Samples (about 100 mg each) were lyophilised using a VD-250R lyophiliser freeze dryer (TAITEC, Saitama, Japan) and ground using a ShakeMaster NEO homogeniser (Bio Medical Science, Tokyo, Japan). Crude DNA was extracted from each group; then, DNA was purified using the MPure-12 Automated Nucleic Acid Purification System (MP Biomedicals, California, USA) with a MPure Bacterial DNA Extraction Kit (MP Biomedicals).
6
Bacterial and Archaeal Community Analysis
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The frozen samples were crashed and for extraction of DNAs using the MPure Bacterial DNA Extraction Kit (MP Bio). The extracted DNAs were quanti ed by Synergy H1 (Bio Tek) and Quanti uor dsDNA System (Promega). The genomic library was then created by using various primers. 16S rRNA genes were ampli ed using the primer 341F (5'-CCTACGGGAGGCAGCAG-3') and modi ed 806R (5'-GACTACHVGGGTATCTAATCC-3') for the bacterial V3V4 region (Muyzer et al. 1999; Caporaso et al. 2011) , the primer modi ed ARC344F (5'-ACGGGGYGCAGCAGGCGCGA-3') and ARC806R (5'-GGACTACVSGGGTATCTAAT-3') for the archaeal V3V4 region (Raskin et al. 1994, Takai and Horikoshi 2000) , and the primer 515f MIX (5'-GTGCCAGCMGCCGCGGTAA-3') and 806r MIX (5'-GGACTACHVGGGTWTCTAAT-3') for the bacterial and archaeal V4 regions (Caporaso et al. 2011) . Two step tailed PCR was then carried out as described previously (Takahashi et al. 2014) . The sequencing was carried out by illumina MiSeq Reagent Kit v3 (2 x 300 bp), and the obtained sequence data were ltered using fastq_barcode_splitter of the Fastx tool kit. High quality rRNA database Silva 128 (Quast et al. 2013 ) was used to construct 97% Operational Taxonomic Units (OTUs) and their phylogenetic estimations. Nucleotide sequence data reported are available in the DDBJ sequenced Read Archive under the accession number DRX221637 to DRX221644 and DRX251432 to DRX251435.
7
Faecal DNA Extraction Protocol
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Faecal samples were collected using a guanidine thiocyanate solution (Faeces Collection kit; Techno Suruga Lab, Shizuoka, Japan). After vigorous mixing, the samples were stored at 4°C for a maximum of 7 days until DNA extraction. After homogenisation with lysis solution F (Nippon Gene, Tokyo, Japan), the genomic DNA was heated at 65°C for 10 min and puri ed from the supernatants using the MPure Bacterial DNA Extraction Kit (MP Biomedicals, Solon, OH, USA) with MPure-12 system (MP Biomedicals).
The puri ed DNA was quanti ed using Synergy LX (BioTek Instruments, Winooski, VT, USA) and QuantiFluor ® system (Promega, Madison, WI, USA).
The puri ed DNA was quanti ed using Synergy LX (BioTek Instruments, Winooski, VT, USA) and QuantiFluor ® system (Promega, Madison, WI, USA).
8
Pollen DNA Extraction and Purification
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First, pollen samples (0.5 g) were lyophilized using a lyophilizer freeze dryer VD-250R (TAITEC, Koshigaya, Saitama, Japan). After being ground at 1500 rpm for 2 min using a ShakeMaster NEO homogeniser (bms, Shinjyuku, Tokyo, Japan), DNA was extracted using the protocol of MPure Bacterial DNA Extraction Kit (MP Biomedicals, Irvine, CA, USA). DNA purification of the samples was performed using the MPure-12 Automated Nucleic Acid Purification System (MP Biomedicals, Irvine, CA, USA). Quality control of DNA extracts was conducted using Synergy H1 (BioTek, Winooski, VT, USA) and QuantiFluor dsDNA System (Promega, Madison, WI, USA).
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