**DNA Extraction Protocol:**
1. Begin by using a 15 mL mortar and pestle. Add 5 mL of blood clot and 4–5 mL of hemolysis reagent. Grind the mixture thoroughly until no visible clots remain. Transfer the resulting suspension into a 50 mL centrifuge tube and centrifuge at 2,500 rpm for 10 minutes.
2. Carefully remove the supernatant and discard it. Wash the pellet once with fresh hemolysis reagent, then centrifuge again at 2,500 rpm for 10 minutes. Repeat this washing step until the supernatant is clear and free of red color.
3. After discarding the supernatant, add 1 mL of cell lysate to the pellet. Transfer the mixture into a new centrifuge tube. Add 10 mL of 20 mg/mL proteinase K and incubate the tube in a water bath at 56°C for 3 hours.
4. Next, add 1 mL of pre-equilibrated phenol to each tube. Vortex the mixture thoroughly and centrifuge at 6,000 rpm for 10 minutes.
5. Repeat step 4 once more to ensure complete separation of the aqueous and organic phases.
6. Carefully transfer the upper aqueous layer to a new 2 mL Eppendorf tube. Add an equal volume of chloroform/isopropanol (2:1) mixture, mix well, and centrifuge at 6,000 rpm for 10 minutes.
7. Take the supernatant from the previous step and transfer it to another 2 mL Eppendorf tube. Add 1/10th volume of 3 M sodium acetate (NaAc), mix, and then add an equal volume of isopropanol. Allow the mixture to sit for 10 minutes, then centrifuge at 10,000 rpm for 5 minutes. A white precipitate should form, indicating DNA isolation.
8. Discard the supernatant, and add 0.3 mL of absolute ethanol to the pellet. Centrifuge at 10,000 rpm for 5 minutes, then carefully remove the ethanol. Let the pellet air dry for a few minutes.
9. Finally, resuspend the DNA pellet in 100–200 µL of TE buffer. Incubate the tube in a water bath until the DNA is fully dissolved. Measure the DNA concentration using a spectrophotometer.
**Important Notes:**
1. Always check the color of the phenol before use. It should be yellow. If it turns pink, it has been oxidized and should not be used.
2. Phenol is highly corrosive and can cause severe burns. In case of skin contact, rinse immediately with water, followed by soap or a diluted baking soda solution. Avoid using alcohol on the affected area.
3. Hemolysis reagent, cell lysate, and TE buffer must be sterilized using high temperature and pressure before use.
4. DNA absorbs ultraviolet light at 260 nm due to its conjugated benzene rings. An A260 value of 1 corresponds to approximately 50 µg/mL of double-stranded DNA. The actual DNA concentration is calculated as: A260 × 50 µg/mL × dilution factor.
5. Purity is assessed by the ratio of A260/A280. Pure dsDNA typically has a ratio around 1.8. A ratio above 1.8 suggests RNA contamination, while a ratio below 1.8 may indicate protein or phenol contamination.
6. Ensure that the DNA is completely dissolved before measuring its concentration. Also, always mix the diluted sample thoroughly to avoid measurement errors.
This protocol ensures a reliable and reproducible method for isolating high-quality genomic DNA from blood samples.
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