HK-2 cells were cultured with or without wogonin for 24 h. After that, cellular proteins were extracted by using RIPA buffer. Adjust the sample to a similar concentration according to the BCA results. Each sample was allocated into multiple PCR tubes and processed at different temperatures for 10 min on a PCR thermal cycler (Eppendorf, Germany). These proteins were then examined by Western blot.
Pcr thermal cycler
Manufactured by Eppendorf
Sourced in Germany
The PCR thermal cycler is a laboratory instrument used for the process of Polymerase Chain Reaction (PCR). It is designed to precisely control the temperature and cycling of samples during the PCR process, which is a fundamental technique in molecular biology and genetics.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Ethidium bromide, by Thermo Fisher Scientific (2 mentions)
Dna extraction kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taq dna polymerase, by Merck Group (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Uv transilluminator, by Vilber (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Cfx connect rt pcr system, by Bio-Rad (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Lightcycler, by Roche (1 mentions)
9 protocols using pcr thermal cycler
1
Wogonin Regulation of HK-2 Cells
2
Identification of Probiotic Marker Genes in L. plantarum
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The presence of probiotic marker genes encoding species-specific collagen-binding protein and bile salt hydrolase was confirmed in LPJBC5, as previously described [34 (link)] (Table S1 ). Additionally, L. plantarum specific sequence and anti-microbial gene (plantaricin-biosynthetic gene) was amplified and sequenced in LPJBC5 (Table S1 ) [35 (link),36 (link)]. The genomic DNA was extracted using a genomic extraction kit, and its concentration was determined using a NanoDropTM 2000c spectrophotometer (Thermo Scientific, USA). The PCR reaction was set up with a total reaction mixture of 25 μL containing 2.5 μL of 10× Taq buffer, 1.5 U of Taq DNA polymerase (Sigma, Darmstadt, Germany), 1.5 mM of MgCl2, 100 μM of dNTP mixture, 10 pmol of each primer pair and 50 ng of bacterial DNA. The PCR was performed using the following conditions in a PCR thermal cycler (Eppendorf, Hamburg, Germany): 96 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min, and 72 °C for 5 min. The amplified product was run on 1.5% agarose gel and imaged under a UV transilluminator (Vilber, Collégien, France). Sequencing of purified PCR amplified fragments was performed with Macrogen Inc. (Seoul, Korea). The DNA sequences were subjected to BLAST analysis and submitted to NCBI (National Center for Biotechnology Information, Bethesda, Rockville, MD, USA) database.
3
Molecular Identification of Streptomyces Using 16S rRNA
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Chromosomal DNA was extracted, and polymerase chain reaction (PCR) was conducted. The template DNA was amplified in an Eppendorf PCR thermal cycler by using a set of published primers of 1 μL of forward (5′-CGCGGCCTATCAGCTTGTTG-3′) and reverse primer (5′- CCGTACTCCCCAGGCGGGG-3′) to target the 16S rRNA gene of Streptomyces (Oloumi et al., 2023 (link)). The amplification was carried out in 30 cycles, denaturation for a minute at 91 °C, primer annealing for 1 min at 56 °C and extension for 5mins at 72 °C. The resulting amplicons were subjected to analysis via 1.5 % agarose gel electrophoresis (Quinn et al., 2020 (link), Saygin et al., 2020 (link)). The amplicon with evidence of electrophoretic bands was sequenced and compared with other pools of organisms using the NCBI BLAST tool for ancestral genomic similarity pairing. A phylogenetic tree was constructed using the neighbor-joining method at a bootstrap of 1000 replicates in MEGA 11 software (Kumar et al., 2016 (link)).
4
Campylobacter DNA Extraction and PCR
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In an anaerobic jar with a gas-generating pouch, frozen Campylobacter isolates were grown on 5% horse blood agar plates and incubated for 48 hours at 42°C. The bacterial cells were taken from the plates and put into Eppendorf vials containing 200 μL of sterile water when they had grown sufficiently. The suspensions were heated for 8 minutes at 98°C on a boiling tube. The supernatant was collected and was then placed into sterile microcentrifuge tubes and centrifuged at 17000 g for 5 minutes to serve as a genetic DNA material for the following polymerase chain reaction (PCR). The quality (A260/A280) and amount of the extracted DNA were next measured at an optical density of 260/280 nm using a spectrophotometer (NanoDrop, Thermo Scientific, Waltham, MA, USA). The DNA's validity was tested on a 1.5% agarose gel stained with ethidium bromide (0.5 g/mL) (Thermo Fisher Scientific, St. Leon-Rot, Germany). The polymerase chain reaction (PCR) was carried out using a PCR thermal cycler (Eppendorf Co., Hamburg, Germany) according to the Tohid and Shandiz technique [18 (link)].
5
Quantitative RT-PCR Analysis of Gene Expression
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The total RNA was extracted by acid-guanidinium-phenol method (Trizol LS Reagent, Invitrogen, USA). RNA was dissolved in 50 μl of RNase-free water. For cDNA synthesis, each 25 µl reaction mixture contained 2 µg of RNA, 0.5 µg of random hexamer primers, 5 µl of 5×RT buffer, 1.25 µl of 10 mml−1 dNTPs, 25 U of RNase inhibitor (Promega), and 200 U of Murine Leukaemia Virus (MLV) Reverse Transcriptase (Promega). cDNA synthesis was performed in a PCR Thermal Cycler (Eppendorf) according to the following procedure: an annealing step for 5 min at 70 °C, followed by reverse transcription for 60 min at 37 °C, and reverse transcriptase inactivation for 10 min at 95 °C. A Light Cycler (Roche) was used for qPCR. The primer sequences are described in Table 1 . Human and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control for THP-1 and Raw264.7 cells, respectively. The Ct was acquired using the CFX Connect RT-PCR system (Bio-Rad, Hercules, CA, USA). All experiments were repeated three times.
6
Identification of Helicobacter Genus by 16S rRNA
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Subculturing was carried out using Wilkins Chalgren anaerobe medium. Genomic DNA was extracted from bacteria using a DNA extraction kit following the manufacturer’s instructions (Cinna-colon, Iran). The extracted DNA’s quality (A260/A280) and quantity (ng/mL) were then assessed using NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). The Helicobacter genus was identified by targeting 16S rRNA (Table 1 ). The oligonucleoide sequence was approved by the Lactofeed Biotech Group (Iran). A PCR thermal cycler (Eppendorf Co., Hamburg, Germany) was used to execute the polymerase chain reaction (PCR) according to the conditions described by the author for the used gene sequences [28 (link)]. Segments of the 16S rRNA gene were analyzed by PCR amplification. PCR conditions were as follows: an initial denaturation (94 °C, 5 min), followed by 40 cycles of denaturation (94 °C, 30 s), annealing (50 °C, 1 min), and extension (72 °C, 2 min), with a final extension (72 °C, 8 min). H. pylori ATCC 700392 strain and distilled water were used as positive and negative controls, respectively.
7
DNA Substrate Preparation and Annealing
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Synthesized DNA oligonucleotides (biomers.net) were resuspended in sterile milliQ water to a concentration of 200 μM and stored at −20 °C. Oligonucleotide sequences used for DNA substrate preparation are listed in Table 3 . DNA annealing was performed in 20 mM HEPES pH 8.0 and 100 mM NaCl. 100 μl reactions including 10 μM of each, the Top and Bottom strand, were heated to 95 °C for 5 min and gradually cooled to 15 °C at a rate of 1 °C min−1 using a PCR thermal cycler (Eppendorf). Annealed DNA substrates were kept at 4 °C in dark storage.
8
H. pylori Genomic DNA Extraction and Verification
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Distinctive colonies of H. pylori were additionally approved using the 16S rRNA-based PCR method. Typical colonies were sub-cultured on Wilkins Chalgren anaerobe broth supplemented with same materials mentioned above [15 (link)]. Genomic DNA was then extracted from colonies using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Procedure was performed rendering to the manufacturer’s guidelines. Purity (A260/A280) and concentration of extracted DNA were then checked (NanoDrop, Thermo Scientific, Waltham, MA, USA). The truth of the DNA was assessed on a 2% agarose gel stained with ethidium bromide (0.5 μg/mL) (Thermo Fisher Scientific, St. Leon-Rot, Germany). Polymerase Chain Reaction (PCR) was performed using a PCR thermal cycler (Eppendorf Co., Hamburg, Germany) according to reported procedure [15 (link)].
9
Molecular Identification of Helicobacter pylori
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H. pylori isolates were additionally confirmed using the 16S rRNA-based PCR method. Colonies were sub-cultured on Wilkins Chalgren anaerobe broth supplemented with the same materials declared above.16 (link),17 (link) Genomic DNA was then extracted using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Technique was performed rendering to the factory guidelines. Purity (A260/A280) and concentration of extracted DNA were then plaid (NanoDrop, Thermo Scientific, Waltham, MA) and the DNA quality was scrutinized by electrophoresis. PCR was accompanied using a PCR thermal cycler (Eppendorf Co., Hamburg, Germany) rendering to the described procedure.18 (link)
H. pylori 26695 was used as positive, while sterile PCR grade water (Thermo Fisher Scientific) was used as negative controls.
H. pylori 26695 was used as positive, while sterile PCR grade water (Thermo Fisher Scientific) was used as negative controls.
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