Fecal samples originating from Nor, Hyp, HySu, and MCT rats were immediately frozen at −80 °C. Total genomic DNA from the samples was extracted. The 16S rRNA genes of the 16S V3–V4 regions were amplified using specific primers with barcodes. PCR was performed with Phusion High-Fidelity Taq Enzyme (NEB) following the manufacturer’s recommendations. The 16S V3–V4 regions were then purified with a DNA Gel Extraction Kit (Promega, Madison, WI, USA) following the manufacturer’s recommendations. An Ion Plus Fragment Library Kit (48 rxns; Thermo Scientific, Waltham, MA, USA) was used to generate sequencing libraries following the manufacturer’s recommendations. Library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific). Finally, the libraries were sequenced on the Ion S5 XL platform, and 400 bp/600 bp single-end reads were generated.
Dna gel extraction kit
Manufactured by Promega
Sourced in United States
The DNA Gel Extraction Kit is a laboratory tool designed to efficiently extract and purify DNA fragments from agarose gels. It provides a simple and reliable method for recovering DNA after electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity taq enzyme, by New England Biolabs (2 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Dna extraction kit, by Qiagen (1 mentions)
Quantifluor st fluorometer, by Promega (1 mentions)
Gcms tq8040, by Shimadzu (1 mentions)
Miseq pe300 platform, by Illumina (1 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (1 mentions)
Xbai restriction endonuclease, by Takara Bio (1 mentions)
4 protocols using dna gel extraction kit
1
Profiling Gut Microbiome in Rodent Models
2
16S rRNA Amplicon Sequencing of Gut Microbiome
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Fecal samples originating from normoxia- and hypoxia-induced MSC-treated mice were immediately frozen at −80°C. Total genomic DNA from the samples was extracted using a QIAGEN DNA extraction kit. The 16S rRNA genes of the 16S V3-V4 regions were amplified using specific primers with barcodes. PCR was carried out with Phusion High-Fidelity Taq Enzyme (NEB) following the manufacturer’s recommendations. Then, the 16S V3-V4 regions were purified with a DNA Gel Extraction Kit (Promega) following the manufacturer’s recommendations. An Ion Plus Fragment Library Kit (48 rxns; Thermo Fisher Scientific) was used to generate sequencing libraries following the manufacturer’s recommendations. Library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific). Finally, the libraries were sequenced on the Ion S5TM XL platform, and 400 bp/600 bp single-end reads were generated. The raw data were deposited in the NCBI public database (accession number: SRR14292713 -SRR14292733 ).
3
Fecal Microbiome and Metabolite Analysis
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Feces samples were stored at −80 °C until further analysis. The Qiagen QIAamp Fast DNA Stool Mini Kit was used in accordance with the manufacturer's instructions to extract DNA samples from the frozen feces and detect them using 1 % agarose. The V3-V4 region of the 16S rRNA gene was amplified using Polymease Chain Reaction (PCR), and its sequence was determined using Illumina high-flux technology by Shanghai Majorboi Bio-pharm Technology Co. Ltd. in Shanghai, China. Prior to sequencing on an Illumina Miseq PE300 platform, the amplicons were purified using an AxyPrep DNA gel extraction kit (Union City, USA), and quantified using a Promega Quantifluor ST fluorometer (Madison, USA).
Feces samples were tested for the presence of SCFAs in accordance with a modified technique (Nakajima et al., 2017 ). The ether layers containing SCFA were collected, combined, and evaluated using a GC/MS-TQ8040 in this study (Shimadzu, Kyoto, Japan). SCFAs concentrations in fecal samples were measured by using the standard external method.
Feces samples were tested for the presence of SCFAs in accordance with a modified technique (Nakajima et al., 2017 ). The ether layers containing SCFA were collected, combined, and evaluated using a GC/MS-TQ8040 in this study (Shimadzu, Kyoto, Japan). SCFAs concentrations in fecal samples were measured by using the standard external method.
4
Construction of Taq DNA Polymerase Chimera
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First, DNA coding sequences of the DNA polymerase from Thermus aquaticus (GenBank: P19821.1) and of CL7, which is a mutant of CE7 from E. coli (GenBank: CP018986.1) were obtained [11] . The two fragments were synthesized by GeneCreate (Wuhan, China). The designed primers for amplification are listed in Table 1.
The gene synthesis procedure was performed as described previously. PCR amplification was then used to obtain two products: the Taq DNA polymerase gene (2493 bp) and the CL7 gene (390 bp). The pET-30 vector was digested with the restriction endonuclease Xba I (Takara, Shiga, Japan), and the product was purified using the DNA Gel Extraction Kit (Promega, Madison, WI, USA). Then the PCR products were mixed together with the digested pET-30 vector. Finally, T5 cloning (New England Biolabs, Ipswich, MA, USA) was performed, in which CL7 was fused to the N-terminus of Taq DNA polymerase with the 7-amino acid linker (ENLYFQG) and a 6 × His tag to the C-terminus. This was necessary for the purification of recombinant protein by metal affinity chromatography. Nucleotide sequences of the resulting recombinant plasmids, pET30/Taq and pET30/CL7-Taq, were confirmed by DNA sequencing (Sangon, Shanghai, China).
Table 1 Primers used in this study for PCR.
The gene synthesis procedure was performed as described previously. PCR amplification was then used to obtain two products: the Taq DNA polymerase gene (2493 bp) and the CL7 gene (390 bp). The pET-30 vector was digested with the restriction endonuclease Xba I (Takara, Shiga, Japan), and the product was purified using the DNA Gel Extraction Kit (Promega, Madison, WI, USA). Then the PCR products were mixed together with the digested pET-30 vector. Finally, T5 cloning (New England Biolabs, Ipswich, MA, USA) was performed, in which CL7 was fused to the N-terminus of Taq DNA polymerase with the 7-amino acid linker (ENLYFQG) and a 6 × His tag to the C-terminus. This was necessary for the purification of recombinant protein by metal affinity chromatography. Nucleotide sequences of the resulting recombinant plasmids, pET30/Taq and pET30/CL7-Taq, were confirmed by DNA sequencing (Sangon, Shanghai, China).
Table 1 Primers used in this study for PCR.
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