DNA Extraction

 DNA extraction is the process of isolating DNA from cells or tissues. DNA can then be used for a variety of applications, such as genetic analysis, cloning, sequencing, and PCR.

There are several methods for DNA extraction, but they generally involve the following steps:

1. Sample collection: The first step in DNA extraction is to collect the biological sample that contains the DNA. This can be blood, saliva, tissue, hair, or any other biological material.
2. Cell lysis: Once the sample is collected, the cells need to be broken open to release the DNA. This can be achieved by physical disruption, such as grinding or homogenization, or by using chemical agents, such as detergents or enzymes.
3. DNA purification: After cell lysis, the DNA needs to be separated from other cellular components, such as proteins, lipids, and RNA. This is typically done using a combination of organic solvents and salts that selectively precipitate the DNA.
4. DNA quantification: Once the DNA is purified, it is important to measure the amount and purity of the DNA. This can be done using spectrophotometry, which measures the absorbance of DNA at a specific wavelength, or by using gel electrophoresis, which separates DNA fragments based on size.

There are many variations of these steps depending on the type of sample and the intended use of the DNA. It is important to use appropriate precautions and sterile techniques to avoid contamination of the DNA during extraction.

Practically Performed:

 Materials:

• Biological sample (e.g. blood, tissue, saliva)
• Cell lysis buffer (e.g. Tris-HCl, EDTA, SDS)
• Proteinase K
• Phenol-chloroform-isoamyl alcohol
• Chloroform
• Isopropanol
• 70% ethanol
• TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)

Equipment:

• Microcentrifuge tubes
• Pipettes
• Vortex mixer
• Heating block or water bath
• Centrifuge
• Spectrophotometer or gel electrophoresis system

Protocol:

1. Collect the biological sample and transfer it to a microcentrifuge tube.
2. Add an appropriate volume of cell lysis buffer and mix well by vortexing.
3. Add proteinase K to the mixture and incubate at 37°C for 30-60 minutes to digest the proteins.
4. Add an equal volume of phenol-chloroform-isoamyl alcohol and mix by vortexing for several minutes to extract the DNA.
5. Centrifuge the mixture at maximum speed for 5-10 minutes to separate the phases.
6. Transfer the upper aqueous phase to a new microcentrifuge tube, avoiding the interface layer.
7. Add an equal volume of chloroform and mix by vortexing to remove residual contaminants.
8. Centrifuge the mixture again and transfer the upper aqueous phase to a new microcentrifuge tube.
9. Add an appropriate volume of isopropanol and mix well to precipitate the DNA.
10. Centrifuge the mixture at maximum speed for 10-15 minutes to collect the DNA pellet.
11. Discard the supernatant and wash the DNA pellet with 70% ethanol.
12. Air-dry the DNA pellet and dissolve it in TE buffer.
13. Measure the concentration and purity of the DNA using a spectrophotometer or gel electrophoresis system.

This protocol is a general guide for DNA extraction and may need to be modified depending on the type and amount of the sample, as well as the downstream applications of the DNA. It is important to follow proper safety precautions and sterile techniques to avoid contamination of the DNA during extraction.