Yeast splice experiment operation method - Database & Sql Blog Articles

This detailed procedure outlines the preparation of competitive RNA for splicing analysis, followed by non-denaturing gel electrophoresis and subsequent blot analysis. The process begins with careful handling of biological samples to ensure minimal contamination. Tissue from mice, specifically small intestine and liver, is collected, flash-frozen in liquid nitrogen, and stored at -70°C. Homogenization is performed using a cold buffer to preserve RNA integrity, and centrifugation steps are used to remove debris and isolate the RNA. RNA extraction involves several purification steps, including phenol-chloroform extraction and ethanol precipitation, ensuring high-quality RNA suitable for downstream applications. After dissolving the RNA in appropriate buffers, it is quantified and checked for purity using spectrophotometry. The final concentration of the competitive RNA is adjusted to between 20–30 μg/μl, meeting the necessary criteria for analytical experiments. Next, the non-denaturing gel is prepared with specific concentrations of acrylamide and other reagents. The gel is poured into a glass plate setup, and after polymerization, it is loaded with the splicing reaction mixtures. Electrophoresis is carried out under controlled conditions, allowing the separation of RNA complexes based on size and charge. The gel is then processed for transfer to a nylon membrane, followed by cross-linking and hybridization with specific probes to detect spliced products. The blotting process includes pre-hybridization, hybridization with labeled probes, and multiple washing steps to remove unbound material. Finally, the membrane is exposed to X-ray film to visualize the results, providing insights into the splicing efficiency and composition of the RNA complexes. This entire protocol ensures accurate and reproducible results, making it a valuable tool in molecular biology research.

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