The isolates were preliminarily assessed through electrophoresis on 1% agarose gel stained with ethidium bromide, with DNA molecular weight ladder as a reference point, as well as by spectrophotometric analysis using Eppendorf UV/VIS BioPhotometer (Eppendorf, Hamburg, Germany). The ratio A260/A280 was used to assess the purity of the isolated DNA and yield.
Uv vis biophotometer
Manufactured by Eppendorf
Sourced in Germany
The Eppendorf UV-VIS Biophotometer is a high-precision spectrophotometer designed for measuring the absorption of ultraviolet and visible light by biological samples. It provides accurate quantification of nucleic acids, proteins, and other biomolecules.
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5 protocols using uv vis biophotometer
1
DNA Purity and Yield Assessment
2
Preparation and Transformation of Competent E. coli
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TOP10 E. coli cells were treated with chemicals (magnesium chloride and calcium chloride) to become competent cells. These chemically competent TOP10 cells were either used directly for transformation or preserved in 15% glycerol stock. IC plasmid was obtained from previously prepared laboratory stock. The transformation of plasmid into competent cells was performed using the heat-shock method. The screening of clones with desired DNA inserts was carried out, and the presence of the IC gene (zot) was checked by performing a standard PCR. IC plasmid was extracted from bacterial clones using NucleoSpin®® PlasmidQuickPure Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The concentration of the purified IC recombinant plasmid was quantified using a UV-VIS Biophotometer (Eppendorf, Germany), and stored at −20 °C as a concentrated stock.
3
RNA Extraction from Liver Tissue
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The mice RNA were extracted from the liver tissues using Total RNA extraction kit (Vivanties Tech., Malaysia) following the manufacturer's instruction. The purity and concentration of the extracted RNA was determined using UV/VIS Biophotometer (Eppendorf, Germany).
4
Apoptotic DNA Extraction and Analysis
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The assay was performed using Apoptotic DNA assay kit (Roche, Germany) following manufacturer's protocol. The DNA was extracted from the liver tissues of treated and untreated mice. Briefly, 200 µl of the cell lysis buffer was added to each 20 mg of liver tissue sample, homogenized and incubated for 10 min at room temperature. Following incubation, 100 µl of isopropanol was added to 100 µl of the clear supernatant in a new tube, vortexed for 60 s and the mixture was transferred to a spin column attached to a collection tube. The tube was centrifuged for 1 min at 8000 rpm, and the flow through was discarded. The DNA pellet trapped in the filter tube was washed by the addition of 500 µl of wash buffer and centrifuged again for 60 s at 8000 rpm. Thereafter, the filter tube was attached to a microcentrifuge tube and DNA was eluted by the addition of 200 µl each of pre-warmed elution buffer. The purity and concentration of the extracted DNA was determined using UV/VIS Biophotometer (Eppendorf, Germany).
Thereafter, the DNA sample collected was mixed with the loading buffer in a ratio of 5:1 and run on 1% agarose gel (stained with Gel red) for 30 min at 80 V. The gel image was captured on an EGel Imager (Life Technologies, UK)
Thereafter, the DNA sample collected was mixed with the loading buffer in a ratio of 5:1 and run on 1% agarose gel (stained with Gel red) for 30 min at 80 V. The gel image was captured on an EGel Imager (Life Technologies, UK)
5
Bacterial Genomic DNA Extraction
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A single bacterial colony from an overnight culture was inoculated into 10 mL of TSB and incubated at 37 °C overnight. Cells were harvested on the following day by centrifuging at 8000× g for 5 min; the supernatant was discarded. A cell pellet was collected and bacterial genomic DNA was extracted using a QIAamp DNA Mini Kit® (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The concentration of the purified genomic DNA was determined using a UV-VIS Biophotometer (Eppendorf, Hamburg, Germany) and stored at −20 °C.
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