General HPV DNA was detected using the GP couple of primers: GP5+ (5′-TTTGTTACTGTGGTAGATACTAC-3′) and GP6+ (5′-GAAAAATAAACTGTAAATCATATTC-3′). The GP6+ primer was biotynilated in order to perform an enzyme-linked assay afterwards. These primers amplify a 150 bp fragment from the L1 region of the HPV genome [32 (link)]. The PCR reaction mix contained 3.5 mM MgCl2, 0.2 mM dNTP mix, 1 μM of each primer, 0.625 U of GoTaq® G2 Hot Start Polymerase (Promega, USA). The total reaction volume was 25 μl with 5 μl of crude DNA extract. PCR was performed in a T100™ Thermal Cycler (Bio-Rad, USA). The amplification protocol used for this PCR was described by Fontaine et al. [33 (link)]. Amplified fragments were visualized under UV light in a 2% agarose gel (Sigma-Aldrich, USA) incubated in 0.5 μg/ml ethidium bromide solution (Sigma-Aldrich).
Ethidium bromide solution
Manufactured by Merck Group
Sourced in United States, Germany
Ethidium bromide solution is a laboratory reagent used in molecular biology and biochemistry. It is a fluorescent dye that intercalates with nucleic acids, allowing for the visualization of DNA and RNA under ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Agarose gel, by Merck Group (4 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Agarose gel, by Promega (3 mentions)
Gotaq g2 hot start polymerase, by Promega (2 mentions)
T100 thermal cycler, by Bio-Rad (2 mentions)
Normal melting agarose, by Merck Group (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Low melting point agarose, by Merck Group (2 mentions)
Bx51 fluorescent microscope, by Olympus (1 mentions)
Smai restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Biorad geldoc 2000, by Bio-Rad (1 mentions)
Synergy microplate reader, by Agilent Technologies (1 mentions)
Nutrient agar broth, by HiMedia (1 mentions)
β nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Eclipse ts100 fluorescence microscope, by Nikon (1 mentions)
27 protocols using ethidium bromide solution
1
HPV DNA Detection Using GP Primers
2
HPV DNA Detection using Consensus PCR
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HPV DNA was detected with a consensus PCR using the primers GP5+ (5’-TTTGTTACTGTGGTAGATACTAC-3’) and GP6+ (5’-GAAAAATAAACTGTAAATCATATTC-3’), amplifying a 150 bp fragment from the L1 region of the HPV genome [29 (link)]. PCR reactions were carried out in 25 μl of a PCR mixture containing 3.5 mM MgCl2, 0.2 mM dNTP mix, 1 μM of each primer, 1 U of GoTaq® G2 Hot Start Polymerase (Promega, USA) and 5 μl of crude DNA extract. PCR was performed in a T100™ Thermal Cycler (Bio-Rad, USA). Amplified fragments were visualized in a 2% agarose gel (Sigma-Aldrich, USA) incubated with 0.5 μg/ml ethidium bromide solution (Sigma-Aldrich). Detection of hr-HPV DNA was performed using an enzyme-linked immunoassay as described by Jacobs et al. [30 (link)] . Oligo-probes for detection of 12 h-HPV genotypes were used (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59).
3
Alkaline Comet Assay for DNA Damage
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The alkaline comet assay was performed with some modification from the previous method (Ooi et al., 2020 (link)). First, the fully frosted slide was pre-coated with 100 µL of 0.6% (w/v) normal melting agarose (NMA; Sigma Aldrich, USA). Then, the harvested WIL2-NS cells were mixed with 0.6% (w/v) low melting point agarose (LMA; Sigma Aldrich, USA) and were pipetted on top of the solidified NMA layer. Once the LMA layer solidified, the slides were gently immersed in the lysis buffer solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton-X) at 4 °C for 12 h. Before electrophoresis, the slide was immersed in the pre-chilled electrophoresis buffer (0.3 N NaOH, 1 mM EDTA) for 20 min to unwind the DNA strands before running the electrophoresis for 20 min. Then, the slide was rinsed with neutralization buffer solution (400 mM Tris) 3 times. After staining with ethidium bromide solution (10 µg/mL; Sigma Aldrich, USA), the slide was covered with a glass slide and being observed by using the Olympus BX51 fluorescent microscope. The individual comet images were analyzed by using Comet Assay IV software.
4
PFGE Analysis of Multidrug-Resistant Staphylococcus aureus
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Pulsed-field gel electrophoresis (PFGE) was performed using a previously described protocol (Pereira et al., 2014 (link)). The chromosomal DNA from MDR and sensitive S. aureus isolates was digested with the Sma I restriction enzyme (Thermo Fisher Scientific Inc., Life Technologies, Grand Island, NY, USA) and subjected to electrophoresis on 1% agarose gels (Promega; Madison, WI, USA) using the following parameters: 200 V (6 v/cm) at 14°C for 21.5 h, with an initial change of 5 s and a final change of 40 s. The gels were stained with 0.5 μg/mL ethidium bromide solution (Sigma-Aldrich, St. Louis, MO, USA) and visualized using a gel imaging system (ChemiDocTM MP System, Biorad, Hercules, CA, USA). The DNA fragment patterns generated by PFGE were analyzed with NTSY-pc software (version 2.0, Applied Biostatistics, Inc., Port Jefferson, NY, USA; Ramazanzadeh et al., 2013 (link)) using the Sørensen–Dice similarity coefficient and the unweighted pair group method with arithmetic mean (UPGMA) clustering approach (Dice, 1945 (link)).
5
Preparation and Storage of 9AA Reagent
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9AA hydrochloride monohydrate (cat #A38401) was purchased from Sigma-Aldrich, St. Louis, MO, USA. A 10 mM stock solution of 9AA was prepared in deionized water, sterile filtered, and stored protected from light at 4 °C. Actinomycin D (cat # 114666) was obtained from Calbiochem (EMD Chemicals, San Diego, CA, USA) and ethidium bromide solution (cat # E1510) was from Sigma.
6
Confirming Liver Cell Origin of Cell Lines
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To demonstrate the liver cell origin of the cell lines, the expression profile of bile duct or hepatocyte-related genes including cytokeratin 7 (CK7), CK19, γ-glutamyl transferase (GGT), α-fetoprotein (AFP), and albumin (ALB) were determined as previously described [35 (link)]. RNA was isolated from the cell line, and cDNA was prepared as mentioned elsewhere [36 (link)]. Alpha-smooth muscle actin (αSMA) primers were used for the exclusion of fibroblast contamination. Primers used in the current experiment are listed in Table S1 .
PCR products were separated in 1.5% agarose gel in Tris-Borate-EDTA (TBE) buffer. A gel was stained with ethidium bromide solution (Sigma-Aldrich, St. Louis, MO, USA) and the images were captured by Bio-Rad Gel Doc 2000 (Bio-Rad, Hercules, CA, USA).
PCR products were separated in 1.5% agarose gel in Tris-Borate-EDTA (TBE) buffer. A gel was stained with ethidium bromide solution (Sigma-Aldrich, St. Louis, MO, USA) and the images were captured by Bio-Rad Gel Doc 2000 (Bio-Rad, Hercules, CA, USA).
7
Phylogenetic Grouping of E. coli Strains
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One hundred seventy-eight UEc strains were cultured in Luria-Bertani (LB; Difco-Becton Dickinson; NJ, USA) broth, and genomic DNA was extracted using a Wizard® Genomic DNA Purification kit (Promega Corporation, WI, USA) according to the manufacturer’ instructions. The amplification of chuA, yjaA, and one genomic fragment (TspE4.C2) was performed on the extracted DNA by multiplex PCR using specific primers (S1 Table ). The PCR products were resolved by electrophoresis on 1.5% agarose gels (Promega Corporation, WI, USA), stained with ethidium bromide solution (Sigma-Aldrich, St. Louis, MO, USA), and visualized with a gel imaging system (ChemiDoc MP System, Bio-Rad; Mexico). The phylogenetic groups were assigned according to the following genotypes: group B2 (chuA+/yjaA+), group D (chuA+/yjaA−), group B1 (chuA−/TspE4.C2+), and group A (chuA−/TspE4.C2−) [16 (link)].
8
Genomic DNA Purity and Integrity
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The genomic DNA purity was assessed according to the mean 260/280 nm ratio in a Synergy™ microplate reader (Biotek, Winooski, VT, USA). The genomic DNA integrity was verified by electrophoresis using a 1% agarose gel (40 mg of analytical grade Agarose, 40 mL of Tris-borate-EDTA buffer) with 0.5 μL of ethidium bromide solution (Sigma-Aldrich/Merck KGaA, Darmstadt, Germany). The DNA bands were visualized using a UV transilluminator.
9
Nanomaterial Synthesis and Characterization
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Ethanol (96%), Petroleum ether (40–60 °C), Dimethyl sulfoxide (DMSO, ≥99.9%) Silver nitrate (AgNO3, ACS reagent, ≥99%), Polyethylene glycol-2000 (PEG-2000), Magnesium nitrate hexahydrate (Mg(NO3)2·•6H2O, ≥98%) 2.5% glutaraldehde, Paraformaldehyde, Anhydrous sodium sulfate (Na2SO4, ≥99%), Anhydrous sodium hydroxide (NaOH, ≥98.0%), 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal bovine serum (FBS), Antibiotic antimycotic solution, phosphate buffer solution (PBS, pH 7.4), Ethylenediaminetetraacetic acid (EDTA), β-Nicotinamide adenine dinucleotide (NADH), Sodium pyruvate (≥99%), Proteinase K, Ethidium bromide solution, Agarose gel, were acquired from Sigma-Aldrich. The Nutrient agar broth was supplied by HiMedia.
10
Agarose Gel Electrophoresis of Polysaccharides
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A 0.88% (w/v) solution of agarose gel was prepared in a TBE buffer (tris, borate, EDTA buffer 1X). For this purpose, 266 mg of agarose were taken and 30 mL of TBE 1X buffer were added, heating in a microwave oven until the agarose was dissolved then proceeded to polymerization, 3 µL of an ethidium bromide solution (10 mg/mL) (Sigma-Aldrich) was added. In order to avoid the formation of bubbles, this solution was gently mixed, and subsequently deposited on the gel until full polymerization. Afterwards, 20 mg of the polysaccharide in the absence of TiO2 were added in lanes 1, 3, and 4, whereas in lane 2 the powders containing the TiO2 scaffolds were placed. Later, a 280 mV voltage was applied for 1 h in the horizontal electrophoresis system and the gel was visualized under ultraviolet light (230 nm wavelength).
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