Yeast splice experiment operation method - Database & Sql Blog Articles

**Preparing Competitive RNA for Splicing Analysis** 1. **Sample Collection and Storage** Begin by wearing dust-free gloves to prevent contamination from RNases. Carefully remove the small intestine and liver from 8–16 mice, one at a time. Avoid touching the gallbladder and pancreas, as they are rich in nucleases. Immediately place the tissues in liquid nitrogen and store them at -70°C for up to one year. 2. **Homogenization** Thaw the small intestines on a clean paper towel at room temperature. Squeeze out any remaining contents and cut the tissue into 5–7 cm pieces. Transfer each piece into a 50 ml sterile centrifuge tube with 10 ml of ice-cold homogenization buffer. The liver can be added directly to the buffer. Ensure that the buffer volume is sufficient to avoid low RNA yield. 3. **Homogenization Process** Before homogenizing, rinse the homogenizer head with DEPC-treated water and ethanol. Homogenize the sample for 30 seconds for small intestine and 1 minute for liver at medium speed. The homogenate should be thick without visible tissue fragments. Allow it to sit at room temperature after processing. 4. **Centrifugation** Transfer the homogenate to a new centrifuge tube and centrifuge at 8500 g for 15 minutes to remove debris. Discard the pellet and collect the supernatant. 5. **RNA Precipitation** Place the supernatant on ice overnight. Centrifuge at 11,000 g for 15 minutes at 4°C to pellet the RNA. Wash the pellet with 70% ethanol and dry it. 6. **RNA Extraction** Dissolve the RNA pellet in lysis buffer and extract using phenol. Separate the aqueous phase and repeat the extraction process. Add isoamyl alcohol to precipitate the RNA again. 7. **Final Precipitation** Add sodium acetate and absolute ethanol to the aqueous phase, incubate at -20°C overnight, and centrifuge. Wash the pellet with 70% ethanol and dry it before dissolving in cold water. 8. **Quantification and Storage** Measure the RNA concentration using a spectrophotometer. Ensure the OD260/OD280 ratio is ≥1.6 for purity. Store the competitive RNA at -70°C for up to two years. **Non-Denaturing Gel Electrophoresis Setup** 1. **Preparation of Glass Plates** Clean all glass plates, combs, and gaskets. Do not silanize the non-notch plate, as it may cause gel detachment during removal. 2. **Gel Casting** Assemble the glass plates and gasket, ensuring the notch aligns properly. Prepare a 3.2% agarose gel with appropriate buffers and pour it into the mold. Insert the comb and allow the gel to polymerize at room temperature. 3. **Electrophoresis Conditions** Place the gel in a cold room and fill the electrophoresis tank with running buffer. Pre-electrophorese at 12.5 mA before loading samples. **Splicing Reaction and Analysis** 1. **Reaction Setup** Mix competitive RNA with R buffer and prepare splicing reactions on ice. Add components such as DTT, potassium phosphate buffer, PEG8000, MgCl2, ATP, and cell extract. 2. **Initiation and Termination** Incubate the reaction at 23°C and add radiolabeled precursor mRNA. Take samples every minute and stop the reaction by adding R buffer and competitive RNA. 3. **Loading and Electrophoresis** Load the samples onto the gel and run at 150–160 V for 14–22 hours. Ensure the gel remains cool during the process. 4. **Gel Removal and Imaging** Remove the gel carefully and expose it to X-ray film at -70°C. For further analysis, treat the gel with methanol and acetic acid, dry it, and transfer RNA to a nylon membrane. **Northern Blot Analysis** 1. **Transfer and Crosslinking** Transfer the RNA to a nylon membrane and crosslink using UV light. This ensures stable binding for hybridization. 2. **Hybridization and Washing** Hybridize the membrane with specific probes, wash thoroughly, and expose to X-ray film to visualize the results. 3. **Multiple Probes and Reuse** The membrane can be reused with different probes after careful deprobing. This allows multiple analyses in a single experiment. This detailed procedure ensures accurate and reproducible results in splicing analysis and RNA detection.

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