20 Maret 2025

Summary

This video provides a step-by-step guide for isolating DNA from blood samples. The process includes preparing the blood with anticoagulants, performing cell lysis, and using a series of centrifugation steps to separate the nucleic acids. The DNA is then precipitated, washed with ethanol, and rehydrated for storage. The protocol highlights the necessary reagents, equipment, and techniques to ensure successful DNA extraction, and it concludes with instructions for proper DNA storage at various temperatures. This clear and concise guide is ideal for laboratory-based DNA isolation procedures.

Takeaways

• 😀 The process begins with collecting 1 ml of whole blood and adding an anticoagulant solution (ETA) to prevent clotting.
• 😀 The blood and anticoagulant mixture is gently mixed to ensure proper blending before being labeled in microcentrifuge tubes.
• 😀 A lysis solution is added to the blood sample, and it is mixed by pipetting multiple times to break down red blood cells.
• 😀 The sample is incubated at room temperature for 10 minutes to improve red blood cell lysis.
• 😀 After incubation, the sample undergoes centrifugation at 13,000 RPM for 1 minute, resulting in a white pellet at the bottom of the tube.
• 😀 The supernatant is carefully discarded, leaving only the white pellet, which is then vortexed briefly to mix.
• 😀 Nucleic acid solution is added to the pellet, followed by pipetting to ensure thorough mixing and further lysis of white blood cells.
• 😀 Protein precipitation solution is added to the sample, and the tube is vortexed for 10-20 seconds to enhance protein removal.
• 😀 The sample undergoes centrifugation again at 13,000 RPM for 3 minutes, resulting in a brown pellet of proteins.
• 😀 Isopropanol is added to the pellet to separate DNA, followed by another round of centrifugation, after which DNA strands appear as white threads.
• 😀 The DNA is washed with 70% ethanol and centrifuged again to remove any remaining contaminants, before being air-dried for 10-15 minutes.
• 😀 Rehydration solution is added to the dried DNA, and the sample is incubated at 65°C for 1 hour or stored at 4°C overnight, or at -20°C for long-term storage.

Q & A

What is the first step in isolating DNA from whole blood?

The first step is to add 1 mL of whole blood (with anticoagulant) into a microcentrifuge tube and gently mix it with the anticoagulant.

Why is it important to use anticoagulants in the blood sample?

Anticoagulants are used to prevent blood from clotting, which ensures the blood remains in a liquid state for the isolation process.

What is the purpose of the lysis solution in this DNA isolation procedure?

The lysis solution helps break open the cells (lysis) to release the DNA and other cellular components into the solution.

How do you ensure that the blood and anticoagulant mix well after adding them to the tube?

You should gently invert the tube several times to ensure that the blood and anticoagulant are well mixed without causing cell damage.

What is the purpose of the protein precipitation solution?

The protein precipitation solution is added to precipitate proteins from the solution, leaving the DNA in the liquid phase for further processing.

Why is it important to centrifuge the samples during the isolation process?

Centrifugation helps separate the components of the blood, such as proteins and DNA, by spinning the sample at high speeds, causing denser materials like the DNA to form a pellet at the bottom of the tube.

What should you do after centrifugation when a white pellet forms at the bottom of the tube?

After centrifugation, carefully remove and discard the supernatant (the liquid layer) without disturbing the white pellet, which contains the DNA.

How is DNA precipitated from the solution?

DNA is precipitated by adding isopropanol, which causes the DNA to form visible strands or threads that can be collected by centrifugation.

Why is 70% ethanol used in the DNA isolation process?

70% ethanol is used to wash the DNA pellet, removing impurities and salts that may interfere with further analysis or storage.

What are the storage recommendations for the isolated DNA?

The isolated DNA should be stored at 2-8°C for short-term storage or at -20°C for long-term storage to preserve its integrity.