Fluorescent proteins were visualized directly in the agarose gels using a UV transilluminator (Cleaver Scientific, Rugby, UK). Blotted membranes were stained with Ponceau S solution, and proteins were also detected by means of anti-HisTag-HRP (horseradish peroxidase) antibodies (Thermo Fisher, Cat No. MA1-80218, Waltham, MA, USA) in combination with Novex™ ECL (Invitrogen Chemiluminescent Substrate Reagent Kit, Cat No. WP20005, Waltham, MA, USA) as a reaction substrate. Membranes were imaged with a UVITEC chemiluminescence imaging system (Cambridge, UK). In situ APEX activity in agarose gels was detected in the presence of TMB (3,3′,5,5′-Tetramethylbenzidine) (VWR—Cat. No. J644-100 ML) until colored bands appeared. APEX was used to identify blotted antigen (SC) by adding ACA diluted 1:200 in PBS/5% milk in combination with a Chemiluminescent Substrate Reagent Kit (Invitrogen, Cat No. WP20005). Membranes were imaged as above. APEX activity was also measured on membranes blotted with ACA. Membrane lanes were cut and incubated with either TMB or Amplex®UltraRed, and APEX activity was measured by using the spectrophotometer to read the absorbance at 450 nm and the fluorescence at 595 nm for TMB and Amplex®UltraRed, respectively.
Uv transilluminator
Manufactured by Cleaver Scientific
Sourced in United Kingdom
A UV transilluminator is a laboratory instrument that uses ultraviolet (UV) light to visualize and analyze DNA or protein samples that have been stained with a fluorescent dye. The core function of a UV transilluminator is to provide a controlled source of UV light that illuminates the stained samples, causing them to emit fluorescent light that can be detected and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Electrophoresis unit, by Cleaver Scientific (2 mentions)
Novex ecl, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
Dntps mix, by Promega (1 mentions)
Taq polymerase, by Promega (1 mentions)
Mgcl2, by Promega (1 mentions)
Loading dye, by Thermo Fisher Scientific (1 mentions)
9 protocols using uv transilluminator
1
Visualizing and Detecting Fluorescent Proteins
2
Agarose Gel Electrophoresis for Genotyping
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Following the restriction endonuclease digestion, DNA fragments were separated using the agarose gel electrophoresis technique at 3% concentration of agarose and the subsequent visualization of bands using a UV transilluminator (Cleaver Scientific, UK). The gel was prepared with ethidium bromide to enhance visualization under UV (Figs 1 and 2 ) . The electrophoresis unit (Cleaver Scientific, UK) was filled with Tris–Borate–EDTA (TBE) buffer, 10 μl of 100 bp molecular ladder (Meridian Bioscience, USA) was loaded and 10 μl of DNA samples were also loaded.
The fragmentation of post restriction endonuclease digestion for Fokl SNP resulted in bands of sizes that depict a participant’s genotype(Fig 1 ) . The presence of only the 272 bp denotes homozygous “FF” genotype, 272 bp and 198 bp denote heterozygous “Ff”, and the presence of only the 198 bp band correspond to homozygous recessive “ff” genotype.
Fragmented Bsml digested by the Bsml restriction enzyme resulted in band sizes that depict the genotypes of participants as homozygous “BB” (825 bp), homozygous recessive “bb” (650 bp and 175 bp) and heterozygous “Bb” (825 bp, 650 bp and 175 bp)(Fig 2 ) .
The fragmentation of post restriction endonuclease digestion for Fokl SNP resulted in bands of sizes that depict a participant’s genotype
Fragmented Bsml digested by the Bsml restriction enzyme resulted in band sizes that depict the genotypes of participants as homozygous “BB” (825 bp), homozygous recessive “bb” (650 bp and 175 bp) and heterozygous “Bb” (825 bp, 650 bp and 175 bp)
3
Blastocystis sp. SSU rRNA Gene Amplification
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The ∼ 620-bp fragment of the barcoding region of the SSU rRNA gene of Blastocystis sp., was amplified using primers RD5 (5’-ATCTGGTTGATCCTGCCAGT-3’) and BhRDr (5’-GAGCTTTTTAACTGCAACAACG-3’) (24 (link)). PCR amplifications were done at standard conditions: an initial denaturing step of 95 °C for 5 min and 35 cycles consisting of 94 °C for 30 s, 58 °C for 30 s, and 30 s at 72 °C. A final extension at 72 °C was performed for 5 min. Finally, 5 μL of amplification products was fractionated on 1.5% agarose electrophoresis gel stained with ethidium bromide and then visualized by the UV transilluminator (Cleaver scientific Ltd., Warwickshire, United Kingdom).
4
Monoplex PCR for Enterotoxin Genes
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The cytoxic enterotoxin (act) and heat-labile enterotoxin (alt) genes were detected by monoplex PCR according to the method of 14 using a pair of primers (Table 1).
The PCR was performed in a final volume of 25 μL containing 4 μL of dNTPs mix (0.2 mM), 4 μL of MgCl 2 (25 mM) (Promega Madison wi USA); 0.2 μM of each primer (IDT, USA); 1.5 μL of Taq polymerase (5U / μL) (Promega, France), 5 μL of 10X PCR buffer, 5 μL of DNA and milliQ water. PCR conditions were done in a Thermocycler (TECHNE) with the following program : an initial denaturation at 95°C for 5min following by 35 cycles consisted of denaturation: 95°C for 15s, annealing: 57°C for 30s (act gene) and 69 °C for 30s (alt gene), extension: 72°C during 30s and a extension at 72°C for 30 following final extension during 10 min at 72°C. PCR products 232bp and 361bp were revealed using a UV transilluminator (Cleaver Scientific LTD) after an electrophoresis in a 1.5% agarose gel in Tris-borate-EDTA (0.5X) (Sigma Aldrich, USA) during 1h at 100 Volt.
The PCR was performed in a final volume of 25 μL containing 4 μL of dNTPs mix (0.2 mM), 4 μL of MgCl 2 (25 mM) (Promega Madison wi USA); 0.2 μM of each primer (IDT, USA); 1.5 μL of Taq polymerase (5U / μL) (Promega, France), 5 μL of 10X PCR buffer, 5 μL of DNA and milliQ water. PCR conditions were done in a Thermocycler (TECHNE) with the following program : an initial denaturation at 95°C for 5min following by 35 cycles consisted of denaturation: 95°C for 15s, annealing: 57°C for 30s (act gene) and 69 °C for 30s (alt gene), extension: 72°C during 30s and a extension at 72°C for 30 following final extension during 10 min at 72°C. PCR products 232bp and 361bp were revealed using a UV transilluminator (Cleaver Scientific LTD) after an electrophoresis in a 1.5% agarose gel in Tris-borate-EDTA (0.5X) (Sigma Aldrich, USA) during 1h at 100 Volt.
5
RNA Extraction and cDNA Synthesis
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Isolation of the total RNA from human whole blood was performed using the PAXGene RNA isolation kit and following the manufacturer instructions. The quality of the total RNA was checked by agarose gel (1.5%) electrophoresis, and the gel was observed with an ultraviolet (UV) transilluminator (Cleaver Scientific, Rugby, Warwickshire, United Kingdom). Complementary deoxyribonucleic acid (cDNA) was synthesised, and its quality was checked with agarose gel (1.5%) electrophoresis with a standardized protocol.
6
Agarose Gel Electrophoresis of DNA Samples
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Agarose gel of 1% concentration was prepared to run the DNA samples. Solidified gel within its casting tray was placed in gel buffer tank (Wealtec Corporation, Model: mini GES), filled with TAE buffer (Cole and Tellez, 2002 (link)). Each DNA sample (5 μl) along with negative control was loaded in the respective wells of the gel. DNA ladder of 100 base pairs (bps), with 13 discrete DNA fragments ranging from 100 to 3,000 bp, was used as the standard to determine the DNA fragment size on gel electrophoresis. After running samples, the gel was taken out from the buffer tank and observed under UV light (UV transilluminator, Cleaver Scientific Ltd.) to visualize the variation among DNA bands.
7
Nucleocapsid Protein Gene Detection
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The cDNA was investigated for the nucleocapsid protein (N) gene using Goldstar PCR Red Master mix (Eurogenetec, Deutschland GmbH, Köln, Germany), oligonucleotide forward primer NP and reverse primer NP4 (Eurofins Genomics GmbH, Ebersberg, Germany) as previously described [35 (link)]. The PCR amplification was performed in a 30 μL reaction mixture with 1 μL (10 pmol μL− 1) of each primer, 15 μL ready-to-use Goldstar PCR Red Master mix and 10 μL Aqua dest. Finally, 3 μL DNA template was added to each reaction tube. The amplification reaction was carried out with the thermocycler program: Initial denaturation at 94 °C for 2 min followed by 35 cycles consisted of 94 °C for 30 s, 55 °C for 60 s, 72 °C for 30 s and final extension 72 °C for 5 min. The amplified PCR products were electrophoresed on 1.5% agarose gel matrix (Biozym Scientific GmbH, Hessisch-Oldendorf, Germany), stained in ethidium bromide (0.5 μg mL− 1) and visualized at 302 nm on a UV transilluminator (Cleaver Scientific Ltd., Warwickshire, UK).
8
Genotyping of IL6 -174G>C Polymorphism
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Genotyping according to the IL6 -174G>C (rs1800795) polymorphism was performed by PCR-RFLP-based methods. In brief, тhe PCR reaction was performed in a mix containing 30 to 50 ng genomic DNA, 0.8 pmol/μl of each primer (IL6-F: 5’-TTG TCA AGA CAT GCC AAG TGC T-3’ and IL6-R: 5’-GCC TGA GAG ACA TCT CCA GTC C-3’), 1х Dream Taq Green PCR Master Mix with 2 mM MgCl2 (2x; Fermentas Life Science) and bdH2O up to the final volume of 12 µl. The temperature profile of the PCR reactions was as follows: denaturing at 95°C for 3 minutes, 35 cycles of denaturation for 30 seconds at 95°C, annealing for 30 seconds at 62°C and polymerization for 30 seconds at 72°C, followed by final elongation at 72°C for 5 minutes.
A volume of 12 µl of the PCR products were digested with 3 U Hin I (Nla III) (Thermo Fisher Scientific Inc.) for 16 hours at 37°C in a mixture of 17 µl. The fragments were analyzed by electrophoresis in 3.5% agarose gel stained with ethidium bromide and detected by a UV transilluminator (Cleaver Scientific Ltd., Rugby, UK).
A volume of 12 µl of the PCR products were digested with 3 U Hin I (Nla III) (Thermo Fisher Scientific Inc.) for 16 hours at 37°C in a mixture of 17 µl. The fragments were analyzed by electrophoresis in 3.5% agarose gel stained with ethidium bromide and detected by a UV transilluminator (Cleaver Scientific Ltd., Rugby, UK).
9
Agarose Gel Electrophoresis for Genetic Variant Analysis
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The process was conducted by transferring the prepared agarose gel onto the gel tank of the electrophoresis unit (Cleaver Scientific, UK). The tank was filled with TBE buffer with ethidium bromide ensuring it covered the gel completely. 10 µl of the ladder (Meridian Bioscience, USA) was loaded and 10 µl of DNA samples were also loaded. For each of the samples, 2 µl of 6X loading dye (ThermoFisher Scientific, UK) was added. The gel was run at 110 V for 45 min. The gel was then visualized under UV transilluminator (Cleaver Scientific, UK) (Fig. 1 ).![]()
Agarose gel electrophoresis of T786C variants (
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