PCR amplification was attempted for the two main types of autotrophic CO2 fixation mechanisms, RubisCO (Calvin-Benson-Bassham cycle) and ATP citrate lyase (reverse TCA cycle) to determine the possible presence of different carbon fixation pathways (Table 1 ). Each PCR reaction contained 1 U of Phire polymerase (Thermo Fisher Scientific, Waltham, MA), 1 X PCR Buffer, 200 μM dNTP, 2 μM each primer, 5 μg BSA, and 1 ng/μl environmental cave DNA. Cycling conditions began with a 2 min hot start at 98°C, followed by 30 cycles of 98°C for 10 s, 60°C for 20 s, and 72°C for 30 s and completed with a 2 min extension at 72°C. PCR product from positive reactions were excised from a 1.5% agarose gel and purified using the GeneJET extraction kit (Thermo Fisher Scientific). Purified PCR products were then ligated into the pJET1.2 vector (Thermo Fisher Scientific) at a 3:1 ratio and transformed into chemically competent cells (Active Motif, Carlsbad, CA). Both strands of inserts from 48 clones were sequenced from each clone library using primers Pjet1.2F and Pjet1.2R. The sequences were then translated and aligned with representative sequences from described isolates and uncultured clones. Phylogenetic trees were calculated using RaxML v7.0.3 (Stamatakis, 2006 (link)) using the Whelan and Goldman model of amino acid substitution (Whelan and Goldman, 2001 (link)).
Genejet extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The GeneJET Extraction Kit is a laboratory product designed for the rapid and efficient extraction of DNA, RNA, or plasmids from a variety of sample types. The kit utilizes a silica-based membrane technology to capture the desired nucleic acids, which can then be eluted for further downstream applications.
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Lab products found in correlation
Phusion hot start 2 high fidelity pcr master mix, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Pjet1.2 vector, by Thermo Fisher Scientific (1 mentions)
Phire polymerase, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Rq manager 1, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sds 2, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Sanger sequencing, by Sangon (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
Premix taq, by Takara Bio (1 mentions)
High fidelity taq polymerase, by Thermo Fisher Scientific (1 mentions)
T4 polynucleotide ligase, by Thermo Fisher Scientific (1 mentions)
High pure plasmid isolation kit, by Roche (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna kit, by Qiagen (1 mentions)
12 protocols using genejet extraction kit
1
Carbon Fixation Pathways in Cave Microbiome
2
Plasmid DNA Extraction and Binding Assay
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pUC18 plasmid DNA was isolated with the GeneJET Plasmid Miniprep Kit (Thermo Scientific). To linearize the plasmid, pUC18 was digested with HindIII and purified with the GeneJET Extraction Kit (Thermo Scientific). Approximately 120 ng of supercoiled or linearized plasmid and increasing concentrations of FLG2 peptide fragments (0.3 μM, 0.9 μM, 1.8μM) were incubated for 30 min at room temperature in 670 mM Tris-HCl, pH 8.8, 160 mM (NH4)2SO4, 25 mM MgCl2, 0.1% Tween20. Samples were loaded immediately onto a 0.8% agarose gel in 0.5 x TAE pH 8.0 buffer, supplemented with 0.05% ethidium bromide, and run at a field strength of 5.7 V/cm. After electrophoresis, bands were visualized under UV light.
3
Nanoparticle-Induced Gene Expression Analysis
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Changes in gene expression were determined by real-time PCR or qPCR, using TaqMan 96-well plates with predesigned assays (4418775, Signal Transduction Pathways) and the Taq-Man Fast Advanced Master Mix (Thermo Fisher Scientific) run on a 7900HT Fast Real-Time PCR System (AB, Thermo Fisher Scientific). Jurkat cells were incubated with the Nps for 24 hours at 100 μg/mL, except for the ZnO Nps where the concentration was 20 times lower (5 μg/mL), and RNA was extracted and purified with the GeneJet extraction kit (Thermo Fisher Scientific). Genomic DNA was removed using DNase I (RNA-free) and complementary DNAs (cDNAs) were synthesized using a Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific). The amount of cDNA per plate and sample was calculated relative to glyceraldehyde 3-phosphate dehydrogenase using different dilutions of cDNA to check the optimal dilution and the qPCR data were analyzed using SDS 2.4 and RQ Manager 1.2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). For each Np, two independent measurements were taken and the value of the relative quantification was averaged, with glyceraldehyde 3-phosphate dehydrogenase used as the internal control. Values with a high standard deviation or genes that were not amplified in one of the two experiments were not taken into account.
4
Sequencing and Analyzing CRISPR Arrays
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PCR-products corresponding to expanded CRISPR array were subjected to high-throughput sequencing with MiSeq Illumina system. The resulting data were analyzed using ShortRead (30 (link)) and BioStrings (31 ) Bioconductor packages. Sequences located between two CRISPR repeats were considered as spacers. They were mapped on phage genomes with no mismatches allowed. R scripts were used for statistical analysis and Circos (32 (link)) was used for graphical representation of the data.
To sequence T7 phage genome in infected cells, total DNA was extracted from infected cells (27 (link)) several minutes before expected lysis time and treated with restriction endonucleases BamHI, EcoRV, HindIII, PstI (do not recognize T7 DNA) to destroy host DNA. Samples were loaded on an 0.8% agarose gel, resolved by electrophoresis and a high-molecular weight band of phage genomic DNA was extracted from gel with GeneJET Extraction Kit (ThermoFisher Scientific) and sequenced with MiSeq Illumina system. Reads were trimmed and mapped on the phage genome with no mismatches allowed.
To sequence T7 phage genome in infected cells, total DNA was extracted from infected cells (27 (link)) several minutes before expected lysis time and treated with restriction endonucleases BamHI, EcoRV, HindIII, PstI (do not recognize T7 DNA) to destroy host DNA. Samples were loaded on an 0.8% agarose gel, resolved by electrophoresis and a high-molecular weight band of phage genomic DNA was extracted from gel with GeneJET Extraction Kit (ThermoFisher Scientific) and sequenced with MiSeq Illumina system. Reads were trimmed and mapped on the phage genome with no mismatches allowed.
5
Detecting Swine Coronaviruses and Rotavirus
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The presence of swine alphacoronaviruses, such as PEDV, Transmissible gastroenteritis virus (TGEV), Swine enteric coronavirus (SeCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV), was tested using a universal primer (Swine Cov F: 5’-AAACTGGAAYTTCASMTGG-3’; Swine Cov R: 5’-ACATARWAAGCCCAWC ) designed by this study. Furthermore, the porcine deltacoronavirus (PDCoV) was detected using a primer set described previously by Sun et al. [26 (link)]. RV VP6 F: 5’-GAAACGGAATAGCTCCACAAT-3’ and RV VP6 R: 5’-GAATAATCAAATCCAGCCACC-3’ are primers. The presence of porcine rotavirus was detected by targeting the VP6 gene with an expected size of 271 bp. Thermo Fisher Scientific’s Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA) was used to amplify the M gene of swine alphacoronavirus and the S gene of PDCoV, which have expected sizes of 547 bp and 1763 bp, respectively. Table 1 lists the primers used in this study. The PCR products were purified using the Gene JET Extraction Kit from Thermo Fisher Scientific (Thermo Fisher Scientific, USA) and then subcloned into the pMD-18T vector (Takara, Japan). Sanger sequencing (Sangon Biotech, China) was used to obtain the M gene sequences, which were then BLAST searched against the GenBank database.
6
Sanger Sequencing Protocol for PCR Products
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For sequence analyses, PCR products were purified from agarose gels by using the MinElute Gel Extraction Kit (Qiagen, Hilden, Germany) or GeneJet Extraction Kit (Thermo Scientific, Waltham, USA) as described in manufacturer’s protocol. For sequencing, a 10 μl mix was used that contained 0.5 μL forward or reverse sequencing primers (25 μM), 4.0 μL BigDye Terminator Cycle Sequencing Kit reagent (Applied Biosystems, Foster City, CA, USA), 4.5 μL H2O and 1 μL DNA. Sequence analysis was performed by using an ABI PrismTM 320 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were further analysis with NCBI BLAST [23 (link)] (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi ) in order to align the obtained sequences with reference sequences.
7
Bacterial Genomic DNA Extraction
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Genomic DNA was purified from bacterial cultures grown overnight in 1 ml of TSB. The genomic DNA GeneJET extraction kit (Thermo Fisher Scientific, Vilnius, Lithuania) was used, following the manufacturer's instructions for Gram-negative bacteria. All DNA was stored at −20°C until further analysis by conventional PCR for virulence genes and clonality by ERIC-PCR.
8
Amplification and Sequencing of Influenza A Virus
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All 8 gene segments of DG/19 were successfully amplified by Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA) with a set of universal primers described by Hoffmann et al. [27 (link)]. The temperature cycle parameters were 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, 53 °C for 30 s, and 72 °C for 2 min, with a final extension at 72 °C for 10 min. The PCR products were purified by using Gene JET Extraction Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Subsequently, the purified PCR segments were incubated for 15 min at 72 °C with Premix Taq (Takara, Japan), then subcloned into the pMD-18 T vector and sequenced by Sanger sequencing.
9
16S rRNA Amplicon Sequencing Protocol
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In short, the V3-V4 regions of the 16S rRNA genes were amplified using the forward primer 341F(5′-CCTAYGGGRBGCASCAG-3′) and the reverse primer 806R(5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were recovered and purified by using GeneJET Extraction Kit (Thermo Fisher Scientific, Wilmington, USA) after gel electrophoresis. Validated libraries were sequenced on the IonS5TMXL platform provided by Personalbio (Shanghai, China). The 16S rRNA gene sequence data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number PRJNA877369 .
10
Standard Molecular Biology Techniques
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Basic DNA manipulations and molecular techniques were done using established procedures [19 ]. Extraction of DNA from agarose gels was done with a GeneJET extraction kit (Fermentas); plasmids were isolated with high pure plasmid isolation kit (Roche). All oligonucleotides used were synthesized at the Unidad de Síntesis of the Instituto de Biotecnología, Universidad Nacional Autónoma de México; all PCR amplifications were carried out using High Fidelity Taq polymerase (Invitrogen). Amplification protocols consisted of 30 cycles of 1 min at 94 °C, 1 min at variable temperature (depending on the primer combination), and 1 to 3 min at 68 °C. After amplification, PCR products were extracted with phenol and precipitated with ethanol. The DNAs were resuspended in Tris-EDTA buffer and digested with the appropriate restriction enzyme(s) to generate the required ends in the fragments. The DNA fragments were purified before cloning by isolating them from the agarose gel. For ligations, T4 polynucleotide ligase (Fermentas) was used. Plasmid transformation of E. coli was done using CaCl2-competent cells. All plasmid constructions were verified by restriction analysis and PCR and, in most of the cases, by DNA sequencing.
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