Using primers 341 F and 806R, amplification of the bacterial 16 S rRNA gene v3-v4 region was performed. PCR testing was then conducted using specific primers with Barcode, Phusion®, and High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs, and high-efficiency high-fidelity enzymes according to the selection of the sequencing region. The amplification procedure was as follows: pre-denaturation at 95℃for 5 min and 35 cycles (denaturation at 95℃for 45 s, retreatment at 55℃ for 45 s, extension at 72℃ for 90 s), followed by stable extension at 72 °C for 7 min, and all PCR products were detected by electrophoresis on a 2% agarose gel. Qualified PCR products were purified by magnetic beads, quantified by enzyme labeling, and samples were mixed in equal amounts according to the concentration of PCR products. After thorough mixing, we used 2% agarose gel electrophoresis to detect the PCR products and used the gel recovery kit provided by Qiagen to recover the products of the target bands. According to the characteristics of the amplified 16 S region, a small fragment library was then constructed, and paired-end sequencing was performed on the library based on the Illumina NovaSeq sequencing platform. Finally, splicing and filtering, OTUs clustering, species annotation, and abundance analysis were all carried out.
Gc buffer
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
GC buffer is a reagent used in molecular biology applications to facilitate the amplification of DNA or RNA sequences with high guanine-cytosine (GC) content. It is designed to improve the efficiency and specificity of the polymerase chain reaction (PCR) or other nucleic acid manipulation techniques by modulating the melting temperature and stability of the target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity pcr master mix, by New England Biolabs (3 mentions)
High fidelity pcr master mix, by New England Biolabs (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Ion plus fragment library kit 48 rxns, by Thermo Fisher Scientific (2 mentions)
Cutsmart buffer, by New England Biolabs (2 mentions)
Phusion hot start flex polymerase, by New England Biolabs (2 mentions)
Gel recovery kit, by Qiagen (1 mentions)
Novaseq sequencing platform, by Illumina (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Truseq dna pcr free sample preparation kit, by Qiagen (1 mentions)
Powersoil dna extraction kit, by Qiagen (1 mentions)
Miseq pe300 platform, by Illumina (1 mentions)
Easypure quick gel extraction kit, by Transgene (1 mentions)
Proteinase inhibitor, by Merck Group (1 mentions)
M cvipi, by New England Biolabs (1 mentions)
Miseq platform, by Illumina (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Exosap, by Thermo Fisher Scientific (1 mentions)
Onetaq hot start 2x master mix, by New England Biolabs (1 mentions)
Onetaq 2x master mix, by New England Biolabs (1 mentions)
22 protocols using gc buffer
1
Amplification and Sequencing of Bacterial 16S rRNA
2
16S rRNA V3 Region Library Preparation
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The library targeting the V3 region of 16S rRNA was constructed by using DNA samples separated from the cecum contents. In brief, individual DNA samples were amplified by PCR using a specific primer with Barcode and Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Beverly, MA). Then PCR amplicon was detected by electrophoresis using 2% agarose gel. The gel extraction kit (Qiagen, GmbH, Germany) was used to isolate the purpose gene fragments. And then, both library concentration and an exact product size were measured using TruSeq DNA PCR-Free Sample Preparation Kit through a quantitative PCR and Qubit. The PCR-free library was constructed based on the illumina Nova sequencing platform. NovaSeq6000 was used for on-board sequencing following qualified the library, and 140 bp paired-end reads were produced, and the raw-sequence data of the whole experiment have been submitted to https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB8069709/overview .
3
Multivector Genetic Manipulation Protocol
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All constructs were PCR-amplified from cDNA using Q5 High-Fidelity DNA Polymerase with GC buffer (NEB). pInducer20 KDM6A WT, MT2 and ΔTPR mutant were previously described [32 (link)]. pLV-EF1A-ACE2 was a gift from Dr. Akiko Iwasaki. PLX307-DPP4 was purchased from Addgene (Cat#158451). For lentiviral transduction, cells were transduced at 50% confluency and selected with puromycin (5 μg/ml) or G418 (1600 μg/ml) 48 hours later.
4
Soil and Root Microbial Community Analysis
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Total DNA was extracted from the rhizosphere soil and root samples according to the instructions of the PowerSoil DNA extraction kit (Mobio Laboratories, Carlsbad, CA, USA). The concentration of the resulting DNA was measured and quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The specific primers 799F (5’-AACMGGATTAGATACCCKG-3´), 1193R (5´-ACGTCATCCCCACCTTCC-3´), and 1392R (5´-ACGGGCGGTGTGTRC-3´), targeted to the V5–V7 regions of 16S rDNA, were used for PCR. PCR was performed using phusion high-fidelity PCR master mix with GC buffer (NEB, Ipswich, MA, USA) and the reaction system and amplification program described [23 (link),24 (link),25 (link)]. The PCR products were detected by agarose gel electrophoresis, and the target DNA band was purified with the EasyPure Quick Gel Extraction Kit (Transgen Biotech, Beijing, China). The purified DNA was submitted to Majorbio Bio-pharm Technology Co., Ltd., (Shanghai, China) for paired-end library construction and sequencing using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).
5
Cloning and Lentiviral Transduction
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All constructs were PCR-amplified from complementary DNA (cDNA) using Q5 High-Fidelity DNA Polymerase with GC buffer (New England Biolabs). For HA- or FLAG-tag HNF1A and HNF1B, the purified fragments were cloned into a lenti-cytomegalovirus vector containing puromycin resistance. pLX307-WT SMARCA4 and its K785R mutant were as described previously34 (link). All constructs were sequence-validated. For lentiviral transduction, cells were transduced at 50% confluency and selected with puromycin 48 h later.
6
16S rDNA Metagenomic Analysis of Ileal Microbiome
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The ileal content from each mice was collected to extract the total bacterial genomic DNA by CTAB method. The V4 hypervariable region of the bacterial 16S rDNA gene was amplified using specific primers with Barcode (515F and 806R), Phusion® High-fidelity PCR Master Mix with GC Buffer (New England Biolabs) and high-fidelity enzyme. TruSeq® DNA PCR-free sample preparation kit was used for library construction. The constructed library was quantified by Qubit and Q-PCR, then sequencing was performed using NovaSeq6000. Pair-end reads was spliced after sequencing according to overlap relation and barcode sequence was removed. Effective sequence was generated after statistical analysis of number and length distribution of sequences, and the percentage of effective sequence was optimized. Sequences with more than 97% similarity were grouped into the operational taxonomic units (OTUs) for subsequent analysis.
7
Methylation of Isolated Nuclei Protocol
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Cell pellet was resuspended with cold lysis buffer: 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) –NaOH pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1× proteinase inhibitor (Sigma 11873580001), 0.1% Tween-20, 0.1 mg/ml bovine serum albumin (BSA), 0.1 mM EDTA (Ethylenediaminetetraacetic acid), 0.5% CA-630, incubate on ice for 5 min. Centrifugate lysis mixture at 500 × g for 5 min at 4°C to collect the nuclei. Washed the nuclei once with 1× GC buffer (NEB M0227L) then resuspend 2 million nuclei in 500 μl methylation reaction mixture: 1× GC buffer, 200 U M.CvipI (NEB M0227L), 96 μM S-adenosylmethionine, 300 mM sucrose, 0.1 mg BSA, 1× proteinase inhibitor, 0.1% Tween-20. Incubate the reaction for 3 hr at 37, add 96 μM SAM (S-Adenosylmethionine) and 20 U M.CvipI per hour. Centrifugate at 500 × g for 10 min at 4°C to collect nuclei, wash the nuclei once with chilled HEPES–NaOH pH 7.5 and centrifugate to collect nuclei.
8
16S rDNA Amplification and Sequencing
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The bacterial genomic DNA was amplified with primers specific for the V4 hypervariable regions of the 16SrDNA gene. The sequences were forward, 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse, 5′-GGACTACHVGGGTWTCTAAT-3′, and the reaction was performed using Phusion High-Fidelity PCR master mix with GC buffer (New England Biolabs, Inc., Ipswich, MA, USA). Samples were sequenced by Illumina MiSeq platform provided by Illumina, Inc. (San Diego, CA, USA) with the following conditions: Denaturation 1 min at 98°C, 10 sec at 98°C, 30 sec at 50°C and 30 sec at 72°Cfor 30 cycles, and 72°C at 5 min.
9
Molecular Cloning of GFP-Gpr Constructs
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Standard molecular cloning techniques were used. All plasmids were derived from pJZ253 (Plac::gfp-gpr in pSRKKm [13 (link)]) by inverse PCR with phosphorylated primers (37 (link)). PCR was performed with the proofreading enzyme Phusion high-fidelity (HF) DNA polymerase and GC buffer (New England Biolabs). PCR conditions were determined empirically for each primer pair. Briefly, forward and reverse primers (20 to 25 mers) were designed to flank the coding sequence for the amino acids to be deleted. Primer sequences were derived from the nucleic acid sequence for gpr (atu1348; GenBank accession number AAK87140 ) and pSRKKm (38 (link)). Each PCR product was then ligated to generate the desired deletion in gfp-gpr. The original stop codon in pJZ253 was retained in all constructs. All constructs were confirmed by DNA sequencing to be in frame with the precise deletion. Resulting plasmids, based on pSRK vectors, placed cloned genes under the control of a tightly regulated lactose-inducible promoter, resulting in expression between 10 and 20% of WT levels (7 (link)). Table S3 in the supplemental material contains additional information on plasmids and strains.
10
Archaea and Eukaryote DNA Extraction
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The DNA in the sample was extracted by the sodium dodecyl sulfate method, and the concentration and purity of the DNA were detected by agarose gel electrophoresis and diluted. Using the diluted genomic DNA as a template, we used foreign objects with Barcode, selected the sequencing area, and used High-Fidelity Polymerase Chain reaction (PCR) Master Mix with GC Buffer (New England Biolabs Phusion®, US) and high-fidelity enzymes for PCR. The amplified regions included the Archaea 16S V4-V5/Archaea 16S V8, 16S V3-V4/16S V4-V5/16SV5-V7; 18S V9, and ITS2 regions.
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