The PCR products were separated by electrophoresis (80 V, 40 min) using a 1% agarose gel (SinaClon, Iran) in 1× Tris/Borate/EDTA buffer. An amount of 5 μl from each of the product was run on the agarose gel. Then, the gel was stained with ethidium bromide (0.5 μg/ml) (SinaClon) and visualized by UV illuminator device (ProteinSimple, USA) and all images were saved on hard disk. A DNA marker of 100 bp (Sinaclon) was used for comparative analysis.
Ethidium bromide
Manufactured by Sinaclon
Sourced in United States, Germany, Iran, Islamic Republic of
Ethidium bromide is a commonly used fluorescent dye for detecting nucleic acids, such as DNA and RNA, in gel electrophoresis applications. It intercalates between the base pairs of nucleic acids, allowing their visualization under ultraviolet light.
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Lab products found in correlation
Agarose gel, by Sinaclon (3 mentions)
Nutrient agar, by Merck Group (1 mentions)
Trypticase soy broth (tsb), by Merck Group (1 mentions)
Pcr master mix, by Ampliqon (1 mentions)
Chef dr 2, by Bio-Rad (1 mentions)
Biodoc it, by Analytik Jena (1 mentions)
Mastercycler, by Eppendorf (1 mentions)
Genegenius gel documentation system, by Syngene (1 mentions)
7 protocols using ethidium bromide
1
Agarose Gel Electrophoresis of PCR Products
2
Shigella ipaH Gene Detection by PCR
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DNA was extracted from Shigella colonies grown overnight on nutrient agar (EMD Millipore) by the boiling method. Briefly, bacteria were cultured in 5 mL of trypticase soy broth (EMD Millipore) and incubated for 24 hours at 37°C. Then, 200 µL of broth culture was mixed with 800 µL of sterile distilled water and the suspension was heated at 95°C for 10 minutes.
Then, the solution was centrifuged at 12,000× g for 5 minutes to remove any cell debris. Finally, 200 µL of the supernatant was stored at –20°C and used as the template for subsequent amplification. PCR amplification was performed to detect the ipaH gene in Shigella isolates.13 (link) Amplification of the ipaH gene was carried out using a thermal gradient cycler (Eppendorf Co., Hamburg, Germany) with the following protocol14 (link),15 : the PCR mixture contained 2.5 mL of 10× buffer (10 mM Tris-HCl and 50 mM KCl), 1.5 mM MgCl2, 3 µL of DNA template, 200 µM each dNTPs, 0.4 µM of each forward and reverse ipaH primer, 0.75 U of Taq polymerase, and sterilized distilled water to complete the reaction volume (25 µL).
The expected sizes of PCR amplicons were revealed by electrophoresis on 1.5% horizontal agarose gel in Tris-borate-EDTA (TBE) buffer and stained with ethidium bromide (0.5 µg/mL) (SinaClon, Tehran, Iran). Primer sequences, PCR conditions, and the PCR product size are shown inTable 1 .16 (link)
Then, the solution was centrifuged at 12,000× g for 5 minutes to remove any cell debris. Finally, 200 µL of the supernatant was stored at –20°C and used as the template for subsequent amplification. PCR amplification was performed to detect the ipaH gene in Shigella isolates.13 (link) Amplification of the ipaH gene was carried out using a thermal gradient cycler (Eppendorf Co., Hamburg, Germany) with the following protocol14 (link),15 : the PCR mixture contained 2.5 mL of 10× buffer (10 mM Tris-HCl and 50 mM KCl), 1.5 mM MgCl2, 3 µL of DNA template, 200 µM each dNTPs, 0.4 µM of each forward and reverse ipaH primer, 0.75 U of Taq polymerase, and sterilized distilled water to complete the reaction volume (25 µL).
The expected sizes of PCR amplicons were revealed by electrophoresis on 1.5% horizontal agarose gel in Tris-borate-EDTA (TBE) buffer and stained with ethidium bromide (0.5 µg/mL) (SinaClon, Tehran, Iran). Primer sequences, PCR conditions, and the PCR product size are shown in
3
Molecular Detection of Legionella pneumophila
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PCR was employed for the amplification of a 159-bp fragment of the mip gene using specific primers (Table 1 ).15 (link) The PCR assay was carried out thus: an initial denaturation at 95°C for 4 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. PCR amplification was performed on an Eppendorf thermocycler 5,530 (Roche). In our study, L. pneumophila serogroup 1 (ATCC 33152) was used as the positive control. Amplicons were separated on 1.5% agarose gel prepared in TAE (Tris–acetate–EDTA) buffer and visualized using the gel-documentation system (ProteinSimple, San Jose, CA, USA) after staining with 0.5 µg/mL ethidium bromide (SinaClon, Tehran, Iran).
4
RAPD-PCR Fingerprinting Protocol for Genomic Analysis
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The primer M13 (GAGGGTGGCGGTTCT) (17) was used for amplification. The PCR reaction was carried out in a total volume of 25 μL, containing 2 μL of primer M13 (10 pmol μL-1), 12.5 μl of 1X PCR Master Mix (Ampliqon, Denmark), 3 μL of genomic DNA (10 ng μL-1), 7.5 μL sterile deionized water. The amplification was carried out in the Eppendorf thermocycler 5530 (Roche, Mannheim, Germany) by using the following program: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 1 min, primer annealing for 1 min at 45°C, and primer extension for 1 min at 72°C with a final extension period at 72°C for 10 min. PCR products were then separated by electrophoresis on 2% (w/v) agarose gel using 1x TBE buffer containing 0.5 μg /mL ethidium bromide (Sinaclon, Tehran, Iran) at 80 V for 2 h. A DNA marker 100bp ladder (Ampliqon, Denmark) was used as a molecular weight standard. RAPD-PCR fingerprints analysis and clustering were processed by the BioNumerics software version 6.6 (Applied Maths, Belgium). Similarities were calculated using the Pearson correlation coefficients. The dendrograms were acquired by means of the clustering algorithm of the Unweighted Pair Group Method using Arithmetic Average (UPGMA).
5
Molecular Typing of MRSE and MRSH
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Typing MRSE and MRSH was performed using the PFGE method as previously described by Talebi et al. [3 (link)]. Briefly, the enzyme digestion of the plugs was carried out with the restriction enzyme SmaI (New England Biolabs). A Salmonella serotype Braenderup strain (H9812) was used as a molecular size marker (kindly provided by the Research Center of Health Reference Laboratories, Tehran, Iran). In addition, DNA separation was carried out by programming two states under the following conditions in a pulsed-field electrophoresis system (CHEF DR-II; Bio-Rad Laboratories, Hercules, CA, USA) at the temperature of 14°C; voltage 6 V/cm; switch angle, 120°C; switch ramp 1-30 second for 20 hours. Ethidium bromide (SinaClon, Iran) 0.5 mg/mL was used for staining gels. For PFGE pattern analysis, BioNumerics software version 7.5 (Applied Maths, St-Martens-Latem, Belgium) was applied. The unweighted pair group method by using mathematical averaging (UPGMA), dice correlation coefficient with 1.5% optimization, and a 1.5% tolerance setting was used for the calculation of dendrograms. Isolates were considered genetically indistinguishable, closely related, possibly related, and different when there were 0, 1, 2, and >3 banding differences, respectively [25 (link)].
6
Plasmid DNA Extraction and Visualization
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EAEC strains grown overnight in Luria broth were used for plasmid DNA extraction by using alkaline lysis method (15 ). Purified plasmid DNA (3 µL) was analyzed by performing gel electrophoresis using 1% agarose gel (SinaClon Co., Iran) stained with ethidium bromide (10 mg/L; SinaClon Co.) and was visualized using a UV transilluminator (BioDoc-It; UVP, USA).
7
Identification of Acinetobacter baumannii by PCR
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From July 2011 to January 2013, a total of 124 non-duplicated A. baumannii isolates were collected from various clinical specimens in two teaching hospitals in Ahvaz, south-est of Iran. Bacterial isolates were initially identified as A. baumannii by biochemical tests (13 ). Suspected isolates were confirmed by PCR to identify blaOXA-51-like gene with specific primers (listed in Table 1 ) to amplify a 353 base pair sequence (14 (link)). DNA template for PCR was obtained by boiling method (15 (link)). Each reaction was carried out in a final volume of 25 µL containing 1x PCR buffer, 1 U Taq polymerase, 1.5 mM MgCl2, 200 µM of dNTP (SinaClon, Iran), 10 pmol of each primer (Eurofins MWG Operon, Germany) and 1 µL of the extracted DNA. PCR conditions were programmed in Mastercycler Eppendorf (Eppendorf, Germany) as follows: Initial denaturation at 94°C for 3 minutes; 35 cycles of 94°C for 45 seconds, annealing 57°C for 45 seconds, extension 72°C for 1 minute and final extension 72°C for 5 minutes. PCR products were separated on 1.5% agarose gel (SinaClon, Iran) by electrophoresis, stained with ethidium bromide (SinaClon, Iran) and then visualized under UV illumination (Syngene GeneGenius gel documentation system). Acinetobacter baumannii ATCC 19606 was used as positive control (14 (link)).
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