PRAKTIKUM DIAGNOSIK MOLEKULER "ISOLASI DNA MENGGUNAKAN PCR"

Summary

This video script demonstrates the process of DNA isolation and PCR installation techniques used in molecular diagnostics. It begins with the importance of isolating DNA from other cellular components and provides a detailed, step-by-step guide on the reagents, equipment, and procedures for DNA extraction, including homogenization, centrifugation, and washing with alcohol. The process continues with PCR preparation, including mixing reagents and DNA templates before running the PCR machine. The video also outlines the final steps, including incubation and results analysis, offering a comprehensive overview of the molecular diagnostic procedure.

Takeaways

• 😀 Introduction to the Molecular Diagnostics group and explanation of DNA isolation using PCI technique.
• 😀 DNA isolation is the process of separating DNA from other cellular components like proteins, lipids, and polysaccharides.
• 😀 The principle of DNA isolation involves breaking down cells to extract DNA, RNA, and other basic substances.
• 😀 Necessary materials for DNA isolation include micropipettes, tips, tubes, tissues, and various chemical solutions.
• 😀 Steps involved in the DNA extraction process, starting with collecting blood and homogenizing it.
• 😀 The first step in DNA extraction is adding 900 microliters of saline solution to the tube, followed by 300 microliters of blood.
• 😀 The sample is then mixed and homogenized, followed by centrifugation at 13,500 RPM for 5 minutes to separate the supernatant.
• 😀 After removing the supernatant, a solution is added, and the mixture is vortexed for 12 seconds to ensure thorough mixing.
• 😀 Isopropanol is added to the mixture to precipitate DNA, which is then centrifuged and washed with ethanol and RNase solution.
• 😀 The final preparation step involves adding DNA template and specific reagents, followed by PCR amplification using a thermal cycler.

Q & A

What is DNA isolation, and why is it important in molecular diagnostics?

DNA isolation is the process of separating DNA from other cellular components such as proteins, lipids, and polysaccharides. It is a crucial step in genetic engineering and molecular diagnostics because it allows researchers to work with pure DNA for further analysis or experimentation.

What is the principle behind the DNA isolation process?

The principle of DNA isolation involves breaking open the cells and extracting their contents, including DNA and RNA. This is followed by purifying the extracted material to obtain a clean DNA sample free from other cellular substances.

What are the key materials and tools used in the DNA isolation process?

The key materials include reagents like lysis solution, DNA rehydration solution, cell lysis solution, protease solution, 70% ethanol, isopropanol, RNase solution, aquades, and a blood sample. Tools used include micropipettes, tips, tubes, and tissues.

Can you explain the initial step in the DNA extraction procedure?

The initial step involves taking a blood sample and transferring it into a tube. The blood is then homogenized to break open the cells and begin the DNA isolation process.

What happens after the blood sample is mixed with the lysis solution?

After adding the lysis solution, the blood sample is mixed in a pipette. This allows the cell membranes to break down, releasing the DNA and other cell contents into the solution.

Why is centrifugation used during the DNA extraction process?

Centrifugation is used to separate the DNA from other cellular components. The sample is spun at high speeds to force heavier particles, like proteins, to settle at the bottom of the tube, leaving the DNA in the supernatant.

What is the purpose of adding isopropanol during DNA extraction?

Isopropanol is added to precipitate the DNA out of the solution. The DNA becomes visible as a stringy, clumped mass, which can then be collected for further analysis.

How is the purity of the DNA sample ensured after precipitation with isopropanol?

After precipitation, the supernatant is discarded, and the DNA is washed with ethanol to remove any residual impurities. The ethanol wash is repeated to ensure a clean DNA sample.

What steps are involved in the preparation of the PCR reaction?

For PCR preparation, several reagents are mixed, including nucleus-free water, Green Master Mix, primary first solution, forward primers, and the DNA template. The mixture is then vortexed to ensure uniformity before being placed in the PCR machine.

What is the final step after setting up the PCR reaction?

The final step is to load the sample into the PCR machine, select the appropriate program based on the group and kit used, and start the amplification process. Once the reaction is complete, the PCR machine is turned off, and the results are analyzed.