Preparation and quantification of eukaryotic DNA

Genomic DNA extraction is a critical step in molecular biology research, especially when studying gene structure and function. The integrity of the extracted DNA is essential, with fragment lengths typically needing to be at least 100–200 kb. To achieve this, it's important to minimize DNA fragmentation and degradation during the process. This ensures that the DNA remains intact for downstream applications such as restriction enzyme digestion, Southern blotting, PCR, library construction, and more. Eukaryotic genomic DNA is usually on the order of 10^7 to 10^9 base pairs and can be isolated from fresh tissue, cultured cells, or cryopreserved samples. A common method involves digesting the sample with proteinase K in the presence of reagents like EDTA and SDS, followed by phenol-chloroform extraction. This approach not only yields high-quality DNA but also ensures its suitability for various molecular techniques. The extraction method may vary depending on the source material, but the general principles remain consistent: avoiding DNase activity and minimizing mechanical shearing of the DNA. These two factors are crucial for maintaining DNA quality throughout the process. **Reagent Preparation:** 1. **TE Buffer**: 10 mM Tris-HCl (pH 7.8), 1 mM EDTA (pH 8.0) 2. **TBS Buffer**: 25 mM Tris-HCl (pH 7.4), 200 mM NaCl, 5 mM KCl 3. **Lysis Buffer**: 250 mM SDS; add 100 mg/ml proteinase K just before use 4. **20% SDS** 5. **2 mg/ml Proteinase K** 6. **Tris-saturated Phenol (pH 8.0), Phenol/Chloroform (1:1), Chloroform** 7. **Absolute Ethanol, 75% Ethanol** **Procedure:** 1. **Sample Handling:** - For fresh or frozen tissue: Cut 0.3–0.5 cm³ of tissue, add 0.5 ml TE, homogenize, transfer to a 1.5 ml tube, add 25 µl of 20% SDS and 25 µl of 2 mg/ml proteinase K, incubate at 60°C for 1–3 hours. - For cultured cells: Wash cells with TBS, centrifuge at 4000g for 5 min, resuspend in 10× lysis buffer, incubate at 50–55°C for 1–2 hours. 2. **DNA Extraction:** - Add equal volume of saturated phenol, mix gently for 3 min, centrifuge at 5000g for 10 min, collect the aqueous phase. - Repeat phenol extraction, then perform phenol/chloroform and chloroform extractions as needed. - Precipitate DNA using 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. Centrifuge, wash with 75% ethanol, dry, and resuspend in TE overnight. 3. **Quantification and Quality Check:** - Measure DNA concentration using a spectrophotometer at 260 nm. A 1 OD₂₆₀ corresponds to approximately 50 µg/ml of double-stranded DNA. - Assess purity using the OD₂₆₀/OD₂₈₀ ratio, ideally around 1.8. A lower ratio suggests protein contamination, while a higher ratio indicates RNA presence. - Run 1 µg of DNA on a 0.8% agarose gel to check for high-molecular-weight bands, indicating good DNA integrity. **Key Considerations:** - All equipment should be sterilized to eliminate DNase. - Use autoclaved water for all solutions. - Handle samples carefully to avoid shearing. - The purified DNA is suitable for most molecular biology experiments, though further purification may be needed for sensitive applications. **Plasmid DNA Extraction via Alkaline Lysis:** Plasmids are small, circular, double-stranded DNA molecules found in bacteria. They replicate independently of the bacterial chromosome and are commonly used as vectors in genetic engineering. Alkaline lysis is a widely used method for isolating plasmid DNA. The process involves lysing bacterial cells with NaOH and SDS, which denatures both chromosomal and plasmid DNA. Upon neutralization, the plasmid DNA renatures and stays in solution, while chromosomal DNA and proteins precipitate out. This allows for the selective recovery of plasmid DNA in the supernatant. Additional purification steps, such as phenol-chloroform extraction, RNase treatment, and ethanol precipitation, can remove residual proteins and RNA. This method is cost-effective and efficient, making it ideal for routine laboratory work such as cloning, transformation, and probe labeling.

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