Bacterial DNA was isolated from fecal samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). The V3-V4 region of the bacteria’s 16S rRNA gene was amplified by thermocycler PCR system (Thermo Fisher Scientific, Wilmington, USA). Amplicons were purified using the DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) and then were quantified. Purified amplicons were sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The 300 bp reads were truncated at any site receiving an average quality score <20. Quality control and splicing of raw data were performed using fastp (version 0.20.0) [37 (link)] and FLASH (version 1.2.7) [38 (link)]. Sequences with 97% similarity were clustered by operational taxonomic units (OTU) using UPARSE software (version 7.1) [39 (link)]. Each sequence was annotated with species classification using an RDP classifier (version 2.2) [40 (link)], and the Silva 16S rRNA database (version 138) was compared, with a 70% threshold. Alpha diversity analysis was used to evaluate the richness and diversity of the samples. The Good’s coverage index was used to reflect the sample coverage. Differences between groups were evaluated using Student’s t-test. Beta diversity was evaluated by unweighted and weighted UniFrac distance based on PCoA analysis.
Thermocycler pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Singapore
The Thermocycler PCR system is a laboratory equipment used for the amplification of DNA or RNA samples through the polymerase chain reaction (PCR) process. It precisely controls the temperature and duration of the various steps involved in the PCR procedure, enabling the replication and amplification of target genetic sequences.
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Lab products found in correlation
E z n a soil dna kit, by Omega Bio-Tek (2 mentions)
Miseq platform, by Illumina (2 mentions)
Tianamp stool dna kit, by Tiangen Biotech (2 mentions)
Axyprep dna gel extraction kit, by Corning (2 mentions)
Quantifluor st, by Promega (2 mentions)
Dna gel extraction kit, by Corning (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Truseq sample preparation kit, by Illumina (1 mentions)
8 protocols using thermocycler pcr system
1
Fecal Bacterial Profiling via 16S rRNA Sequencing
2
Microbial Community Profiling via 16S rRNA
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Microbial DNA was extracted from four groups of samples (day 0, day 6, day 10) using the E.Z.N.A. soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.). The DNA quality was checked by 1% agarose gel electrophoresis. The diluted-genomic DNA was used as a template, and the V3–V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers by the thermocycler PCR system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Each sample was subjected to three replicate experiments. The reference point was subjected to preliminary quantification and the PCR product was quantified by QuantiFlour TM-ST Blue Fluorescence System (Promega) and then mixed according to the sequencing amount of each sample. According to the standard procedure of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China), the combined equimolar purified amplicons were paired-end sequenced on an Illumina MiSeq platform.
3
16S rRNA Amplicon Sequencing Protocol
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The extracted DNA from each sample was used as the template to amplify the V3-V4 region of 16 s rRNA genes with bar-coded primers, forward primers-338F (5′- sampleIDtag- ACTCCTACGGGAGGCAGCA-3′) and reverse primers-806R (5′- sampleIDtag-GGACTACHVGGGTWTCTAAT). PCR reactions were run in a thermocycler PCR system (Applied Biosystems, USA) using the following programme: 5 min of denaturation at 96 °C followed by 25 cycles of for 30 sec at 96 °C (denaturation), 30 sec at 50 °C (annealing) and 30 sec at 72 °C (elongation), with a final extension at 72 °C for 5 min. PCR product was excised from a 1.5% agarose gel and purified by QIAquick Gel Extraction Kit (QIAGEN, cat# 28706). Purified PCR products from the fecal samples were combined at equal concentrations and used to construct a metagenomic library using an Illumina TruSeq sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Barcoded V3-V4 amplicons were sequenced using a pair-end method via the Illumina Miseq (Illumina, San Diego, CA, USA) sequencing platform with a 6-cycle index read.
4
Endophytic Bacteria 16S rDNA Sequencing
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The endophytic bacteria DNA was extracted by the methods of Zhou et al. (2010) (link). Then the 16S rDNA sequences of endophytic bacterial isolates were amplified with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) by a thermocycler PCR system (Applied Biosystems, Singapore) (Mao et al., 2012 (link)). The PCR reaction was performed according to the method of Wang et al. (2019) (link). The PCR products were checked on 1.2% agarose gel and were sequenced using the dideoxy chain-termination method. The two overlapping sequence reads of each endophytic bacterial isolate were assembled by the SeqMan of DNASTAR.Lasergene.7.1, then compared by the 16S-based ID of EzBioCloud (2018) database, and submitted to NCBI Genbank. The phylogenetic tree was constructed by the neighbor-joining method through the MEGA7.0 software.
5
Intestinal Microbiome DNA Extraction and Sequencing
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Intestinal DNA was extracted using a TIANamp Stool DNA Kit (TIANGEN Biotech), according to the manufacturer’s instructions. The final DNA concentration and purification were determined using a Nano Drop 2000 UV-vis spectrophotometer (Thermo Scientific), and DNA quality was checked by 1.5% agarose gel electrophoresis. The V3-V4 hyper-variable regions of the bacterial 16S rRNA gene were amplified with primers 338-F (5′-ACTCCTACGGGAGGCAGCAG -3′) and 806-R (5′-GGACTACHVGGGTWTCTAAT-3′) by the thermocycler PCR system (Applied Biosystems). The PCRs were implemented using the following steps: 3 min of denaturation at 95°C, 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, 45 s for elongation at 72°C, and a final extension at 72°C for 10 min. PCR was performed in triplicate in a 20-μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase, and 10 ng of template DNA. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) and then quantified using QuantiFluor-ST (Promega, Madison), according to the manufacturer's protocol.
6
Endophytic Microbial DNA Extraction and Analysis
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The endophytic microbial DNA was extracted following the standard procedure46 , with slight modifications. Bacterial 16S rRNA genes were amplified using the genomic DNA as template and bacterial universal primers of 27 f and 1492r by thermocycler PCR system (Applied Biosystems, Singapore). The PCR reaction were performed in 50 μL mixture containing 25 μL of 1 × EasyTap PCR SuperMix (TransGen Biotech), 1.5 μL of universal primers 27 f, 1.5 μL of universal primers 1492r, 20 μL of sterilized water, 2 μL of genomic DNA. The PCR amplification procedure was shown as follow (Supplementary Table S3 ). The PCR products were checked for the expected size on 1.2% agarose gel and were sequenced at Sangon Lab (Shanghai, China) with the two primers.
The EzTaxon database (https://www.ezbiocloud.net/ ) was used to compare the 16S rRNA gene sequences of the endophytic bacterial strains, and the sequences were submitted to NCBI Genbank. The phylogenetic tree was constructed using the Neighbour-Joining method through the Mega7 software.
The EzTaxon database (
7
Rapid SNP Genotyping via T-ARMS-PCR
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Tetra-amplification refractory mutation system–polymerase chain reaction (T-ARMS-PCR), which is a rapid and simple technique for studying of SNPs (Ye et al., 2001), was conducted for allele and genotype detection. The primers used for the position was designed using oligo7 and were blasted in NCBI website: http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Table 2 ). Two allele-specific (inner) primers have been designed in opposed directions and, in combination with the common primers, can simultaneously amplify both the mutant allele and wild type alleles in a single-tube PCR. PCR-amplified DNA samples were electrophoresed in agarose gel (2%). After that, 3 amplified samples were examined by DNA sequencing in order to validate the results determined by T-ARMS-PCR.
Genotyping was performed by T-ARMS PCR using the Taq-PCR Master Mix (Cat. No: 5200350-0050, Lot. No: 13C18, Amplicon, Denmark) and thermocycler PCR System (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained a total volume of 20μl (master mix 10μl, forward and reverse inner primer 1μl each, forward and reverse outer primer 0.2μl each, and H2O 7.6μl). The thermocycler conditions were: 95°C for 5 minutes, then 35 cycles of 95°C for 30 seconds 56°C for 30 seconds, and 72°C 40 seconds and final extension of 72°C for 10 minutes.
Genotyping was performed by T-ARMS PCR using the Taq-PCR Master Mix (Cat. No: 5200350-0050, Lot. No: 13C18, Amplicon, Denmark) and thermocycler PCR System (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained a total volume of 20μl (master mix 10μl, forward and reverse inner primer 1μl each, forward and reverse outer primer 0.2μl each, and H2O 7.6μl). The thermocycler conditions were: 95°C for 5 minutes, then 35 cycles of 95°C for 30 seconds 56°C for 30 seconds, and 72°C 40 seconds and final extension of 72°C for 10 minutes.
8
Intestinal Microbiome DNA Extraction and Sequencing
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Intestinal DNA was extracted using a TIANamp Stool DNA Kit (TIANGEN Biotech), according to the manufacturer’s instructions. The final DNA concentration and purification were determined using a Nano Drop 2000 UV-vis spectrophotometer (Thermo Scientific), and DNA quality was checked by 1.5% agarose gel electrophoresis. The V3-V4 hyper-variable regions of the bacterial 16S rRNA gene were amplified with primers 338-F (5′-ACTCCTACGGGAGGCAGCAG -3′) and 806-R (5′-GGACTACHVGGGTWTCTAAT-3′) by the thermocycler PCR system (Applied Biosystems). The PCRs were implemented using the following steps: 3 min of denaturation at 95°C, 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, 45 s for elongation at 72°C, and a final extension at 72°C for 10 min. PCR was performed in triplicate in a 20-μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase, and 10 ng of template DNA. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) and then quantified using QuantiFluor-ST (Promega, Madison), according to the manufacturer's protocol.
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