DNA isolation from fish.

Summary

In this video, Pallavi from Amplicons of Biotech walks viewers through the process of DNA extraction from fish fins, specifically using tilapia. She explains the purpose of DNA isolation, including studying genetic diseases, diagnostics, sequencing, and detecting viruses. The step-by-step procedure includes tissue preparation, homogenization, adding lysis buffer, proteinase K treatment, phenol-chloroform extraction, DNA precipitation with ethanol, and confirmation through agarose electrophoresis. The video concludes with a preview of an upcoming tutorial on agarose electrophoresis, inviting viewers to like, share, and subscribe.

Takeaways

• 😀 DNA is a hereditary material found in humans, plants, animals, and almost all organisms.
• 😀 DNA extraction is essential for studying genetic causes of diseases, developing diagnostics and drugs, sequencing genomes, detecting bacteria and viruses, and determining paternity.
• 😀 The DNA isolation process starts with cutting a small piece of fish dorsal fin (from tilapia) for analysis.
• 😀 The fin sample is washed and dried on tissue paper before cutting into smaller pieces for further processing.
• 😀 The sample is transferred to a micro centrifuge tube, and lysis buffer (containing Tris HCl, SDS, and EDTA) is added to break down the cell membrane.
• 😀 Homogenization is done using a homogenizer to finely crush the tissue sample.
• 😀 Proteinase K is added to the sample to break down proteins, and the sample is incubated overnight at 55°C for optimal results.
• 😀 After incubation, phenol-chloroform extraction is used to purify the DNA, which involves mixing and centrifuging to separate the DNA from other substances.
• 😀 Ethanol is added to precipitate the DNA, which is then collected as a white pellet at the bottom of the tube after centrifugation.
• 😀 The DNA pellet is air-dried to remove any ethanol residues, and then nuclease-free water is added to dissolve the DNA for storage at -20°C.
• 😀 To confirm the presence of DNA, agarose gel electrophoresis is performed, and the results are visualized under UV light to reveal the amplicons as white bands.

Q & A

What is DNA, and why is it important to isolate it?

DNA (deoxyribonucleic acid) is the hereditary material in nearly all organisms, including humans, plants, and animals. It is important to isolate DNA for studying genetic causes of diseases, developing diagnostics and drugs, sequencing genomes, detecting bacteria and viruses, and determining paternity.

What is the purpose of using tilapia fish for DNA isolation?

Tilapia fish is used as the sample for DNA extraction in this procedure. Fish fins, particularly from the dorsal side, are chosen because they provide a sufficient amount of tissue for DNA isolation.

Why is it necessary to cut a small piece of the fish's dorsal fin?

A small piece of the fish’s dorsal fin is cut to provide the tissue needed for DNA extraction. The dorsal fin is chosen because it contains enough genetic material and is easily accessible for sampling.

What is the function of the lysis buffer in DNA extraction?

Lysis buffer is used to break down the cell membranes and dissolve the cellular contents, including the DNA, enabling its extraction. It contains chemical components such as Tris-HCl, SDS, and EDTA that help to dissolve the cellular components.

What does the homogenizer do in the DNA extraction process?

The homogenizer is used to crush the tissue sample into a fine, uniform mixture, ensuring the efficient breakdown of cells for DNA isolation.

Why is Proteinase K added during the extraction process?

Proteinase K is added to the sample to digest proteins that may interfere with DNA isolation. This enzyme breaks down the proteins, ensuring that only DNA is left for further processing.

Why is the sample incubated overnight at 55°C?

The sample is incubated overnight at 55°C to allow Proteinase K to effectively digest the proteins and break down the cellular structures, aiding in the release of DNA.

What is the purpose of phenol-chloroform extraction in DNA isolation?

Phenol-chloroform extraction is used to separate the DNA from other cellular debris, proteins, and lipids. It helps to purify the DNA by removing contaminants before precipitation.

Why is absolute ethanol added during the DNA extraction process?

Absolute ethanol is added to the sample to precipitate the DNA. It helps in separating the DNA from the solution, allowing it to form a visible pellet at the bottom of the tube.

How is the DNA confirmed after isolation?

The DNA is confirmed through agarose gel electrophoresis, a technique that separates DNA fragments based on size. The results can be visualized under UV light, where the DNA will appear as distinct bands.