Polymerase chain reaction (PCR) was carried out as described previously [1 (link)]. In brief, a volume of 50 µL containing 1 µM of each primer, 1 µL of genomic DNA (approximately 200 ng), 1 mM of dNTPs mix (1 µL), 0.5 µL Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA) were used to detect the presence of H. pylori cagA and vacA s and m regions. PCR amplifications were carried out using Applied Biosystems Thermal PCR Cycler (Thermo Fisher, USA). Table 1 summarized the details of the primers sequences used in this study and Figure 1 showed the PCR results of the amplified genes. After optimization, the following cycle conditions were used for cagA: 35 cycles of 7 s at 98 °C, 30 s at 56 °C, and 30 s at 72 °C, while, the following conditions were used for all vacA primers: 35 cycles of 7 s at 98 °C, 30 s at 53 °C, and 30 s at 72 °C. Negative controls and positive samples were included in each run. Amplified PCR products were then resolved by electrophoresis on 2% agarose gels run and visualized using Thermo Fisher Scientific gel documentation system.
Gel documentation system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The gel documentation system is a laboratory instrument designed to capture and analyze images of electrophoresis gels, such as those used in DNA, RNA, or protein analysis. The system typically includes a camera, light source, and software to acquire, process, and store the images.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitors cocktail, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Brain derived neurotrophic factor (bdnf), by Alomone (1 mentions)
Paraformaldehyde (pfa), by Maclin Biochemical Technology (1 mentions)
Sds page system, by Merck Group (1 mentions)
Thermocycler, by Thermo Fisher Scientific (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Dna extraction kit, by Qiagen (1 mentions)
Sabouraud dextrose broth (sdb), by HiMedia (1 mentions)
Sabouraud dextrose agar, by HiMedia (1 mentions)
Thermocycler, by Eppendorf (1 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
Emeraldamp max pcr master mix, by Takara Bio (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Oligonucleotide primers sequences, by Metabion (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Nupage electrophoresis system, by Thermo Fisher Scientific (1 mentions)
10 protocols using gel documentation system
1
Molecular Detection of H. pylori Virulence Factors
2
BDNF-Induced Protein Analysis in DIV Neurons
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Seven DIV neurons, plated at a density of 106 cells/cm2, were incubated in B27-free neurobasal medium for 1 h and then treated with 50 ng/mL of BDNF (Alomone). After washing, neurons were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, and 0.1% SDS) and supplemented with protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktail (Roche, Basel, Switzerland). The homogenates were cleared by centrifugation at 14.000 rpm for 10 min. Protein extracts (50–70 µg) were loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes (Fisher Thermo Scientific). The membranes were blocked with 3% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies. After incubation with the appropriate HRP-conjugated secondary antibody (1:3000, Thermo Scientific), membranes were incubated with ECL substrate for detection of HRP enzyme activity (Thermo Fisher Scientific) and visualized in a Syngene gel documentation system. Images were quantified by ImageJ analysis (National Institutes of Health). 2–4 biological replicates were performed for every experiment. The number of repetitions for every experiment is stated in the corresponding figure description. The Student’s t-test was used for statistical significance assessment.
3
OSCC Cell Proliferation Assay
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After abovementioned transfection, OSCC cell proliferation was evaluated using colony formation assay. Cal27 and SCC9 cells were grown in the culture plate (3 × 103/100 µl) in an incubator for another 24 h at 37 °C containing 5% CO2. Every two days, the culture medium was changed. After 14 days, the incubation was terminated. Cells were purified twice with phosphate buffered saline (PBS) (Suntip, Guangdong, China) after removing the solution. Colonies were fixed with 5% paraformaldehyde (Macklin, Shanghai, China) and then stained with 1 ml of 0.1% crystal violet solution (Le Heng Technology, Shenyang, China). After PBS-washing, the colony number was counted using a gel documentation system (Thermo Fisher Scientific).
4
SDS-PAGE Protein Separation Protocol
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The expression of the protein samples were studied using the SDS-PAGE system (Sigma-Aldrich, St. Louis, CA, USA). There were two separate gels (4% stacking gel and a 12% resolving gel) that were used to isolate the proteins (Table 1 ). In each well, an equal amount (12 µL) of each sample was filled while 5 µL of protein ladder (fermentase) (Thermo Fisher Scientific, Bradford, MA, USA) with molecular weights ranging around 10 to 200 kDa were loaded in one of the wells. The gel was run at 120 volts for 120 min in the electrophoresis tank. The bands in each well were observed by soaking the gel for 40 min in a 50 mL of staining solution. The gel was placed in a destining solution for 5 h, and clear bands were identified using the gel documentation process (Thermo Fisher Scientific gel documentation system).
5
Amplifying Diverse Gene Families by PCR
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Primers from different gene families, such as ascorbate peroxidase (APX), myeloblastosis (MYB), and zinc finger protein (Zat-12) were used to amplify cDNA in a thermocycler (Applied Bio systems, Foster city, CA, USA). The PCR conditions were as follows: (pre-denaturation at 95 °C for 5 min, 30 cycles, (95 °C for 20 s), (60 °C for 30 s) and (72 °C for 4 s) and the final temperature was (72 °C for 12 min). The samples were then run on a 1.5% agarose gel. e. Using a gel documentation method, the difference in the bands was observed (Thermo Fisher Scientific gel documentation system, MA, USA).
6
Visualizing Fungal Colonization and Detecting Trypophobia in Soybean Roots
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Fungal colonization was visualized in roots cut into 1 cm segments (Zhang et al., 2012 (link)). Root samples were first immersed in 10% (w/v) KOH for 2 h at 90°C before being stained with trypan blue. Root pieces were mounted on plates using polyvinyl alcohol and glycerol and viewed under a digital microscope at 20× magnification.
The molecular detection of TH in soybean roots was performed as previously described (Horn et al., 2016 (link)). Briefly, DNA was extracted from soybean roots using Wizard Genomic DNA Purification Kits (Promega Corporation) per the manufacturer's protocol. DNA was then quantified using a NanoDrop ND‐1000 Spectrophotometer (Wilmington) and used for PCR amplification with primers designed to amplify a fragment of the UAOX1 gene of TH. The UAOX1 forward (5′‐GTC GGT AGC TGA AAG GGG AT‐3′) and reverse (5′‐ATG TAG AGG CCG GAA ACA CC‐3′) primer combinations (Horn et al., 2016 (link)) resulted in a 486‐bp fragment from a chromosomal location just upstream of the UAOX1 coding region. The PCR product was then visualized in a Gel Documentation System (Thermo Fisher Scientific).
The molecular detection of TH in soybean roots was performed as previously described (Horn et al., 2016 (link)). Briefly, DNA was extracted from soybean roots using Wizard Genomic DNA Purification Kits (Promega Corporation) per the manufacturer's protocol. DNA was then quantified using a NanoDrop ND‐1000 Spectrophotometer (Wilmington) and used for PCR amplification with primers designed to amplify a fragment of the UAOX1 gene of TH. The UAOX1 forward (5′‐GTC GGT AGC TGA AAG GGG AT‐3′) and reverse (5′‐ATG TAG AGG CCG GAA ACA CC‐3′) primer combinations (Horn et al., 2016 (link)) resulted in a 486‐bp fragment from a chromosomal location just upstream of the UAOX1 coding region. The PCR product was then visualized in a Gel Documentation System (Thermo Fisher Scientific).
7
Standardized Antifungal Susceptibility Assay
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For the microbiological processing and antifungal studies, Sabouraud Dextrose Broth (SDB), Sabouraud dextrose agar, and HiMedia Differential Chromium Agar were obtained from Hi-Media labs, Mumbai. For molecular experiments, a DNA extraction kit was obtained from Qiagen. For polymerase chain reaction (PCR) amplification and amplicon determination, the Eppendorf thermocycler (Germany) and gel documentation system from ThermoFischer were used, respectively. The solvents for extraction, such as DMSO, were obtained from Sudhakar Biologicals, Chennai.
8
Molecular Detection of Mycobacterial Genes
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PCR was used to detect the IS901 gene to confirm the diagnosis of the recovered isolates as well as to investigate the presence of inhA, rpoB, rpsL, and otrB genes in the recovered isolates. Extraction of DNA was performed using the QIAamp DNA Mini Kit (Qiagen, GmbH, Germany/ Catalogue No.51305). The PCR reaction performed in a “25- μl” reaction volume containing; “12.5 μl” of Emerald Amp Max PCR Master Mix (Takara, Japan), one μl of each primer of 20 pmol concentration, 4.5 μl of water, and 6 μl of DNA template. The oligonucleotide primers sequences (Metabion International AG, Germany) and their recycling conditions are illustrated in Table 1 [35 (link)–39 (link)]. Positive control strains (kindly provided by A.H.R.I, Egypt) and negative controls (DNA-free) were involved in each PCR run. Finally, the agar gel electrophoresis was carried out using 1.5% agarose stained with ethidium bromide 0.5 μg/ml (Fermentas, Germany). The gel was visualized by a gel documentation system (Thermo Fisher Scientific, Waltham, MA, USA).
9
Soil DNA Extraction and Quantification
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Two soil samples were collected in triplicate and pooled together for each sample. DNA extraction was done using Powersoil® DNA Isolation Kit (Qiagen, USA) following the manufacturer’s instructions. The metagenomic DNA was checked for integrity by agarose gel (1%) using a BioRad Gel documentation system and was quantified by Qubit 3.0 Fluorometer (Invitrogen, USA).
10
Cell Lysis and Protein Analysis
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Cells were plated at density of 4.8×104 cells per well in 6-well plates and treated with vehicle or drugs. After the indicated time of treatment, cells were lysed by brief sonication in whole-cell lysis buffer (62.5 mM TRIS, 10% glycerol, 1% SDS, pH 6.8), and cellular proteins were collected in the supernatant fraction after centrifugation at 14,000 g for 10 minutes. Proteins were reduced by heating at 100°C for 5 minutes in 50 mmol/L DTT with 0.05 bromphenol blue, electrophoresed in 4–20% Tris-HCl gels, and transferred onto nitrocellulose using the NuPage electrophoresis system (Invitrogen, Carlsbad, CA). The membranes were probed with antibodies to LC3 and cleaved caspase 3 (Cell Signaling, Danvers, MA USA) and actin (Santa Cruz Biotechnology Company, Santa Cruz, CA). Membranes were treated with appropriate secondary antibody, exposed to enhanced chemiluminescence solutions (Invitrogen, Carlsbad, CA) and visualized using the Molecular Imager Gel Documentation System.
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