Chapter 10: DNA Extraction

Chapter 10: DNA Extraction

# 1. Introduction

DNA extraction is the process of isolating DNA from cells or tissues, removing unwanted components to obtain pure DNA. The process involves the following main steps:

# 2. Steps in DNA Extraction

2.1. Removing the nuclear membrane and cell membrane

• For plants: Crush cells by grinding or using liquid nitrogen.

• Using a mixture of SDS and proteinase: SDS breaks down the cell membrane and nuclear membrane, proteinase K breaks down the proteins bound to DNA.

• Stabilize with TE buffer (pH = 8): TE buffer helps maintain a stable pH for the extraction process.

2.2. Removing impurities

• Proteinase K: Continue to remove proteins.

• CTAB: Remove polysaccharides and lipids.

• Chloroform: Separate and pull non-DNA components down due to the difference in density.

2.3. DNA precipitation

• Using 96% ethanol (2:1 ratio) or isopropanol (1:1 ratio): DNA will precipitate and can be easily recovered.

2.4. Washing and storing DNA

• Wash with 70% ethanol: Remove excess salt and isopropanol.

# 3. Properties of DNA

• DNA absorbs ultraviolet light at 260 nm: Due to the structure of the bases.

• Pure DNA: Has an OD260/280 ratio of 1.8 to 2.

• Corresponding OD concentration: 50 µg/ml for double-stranded DNA and 40 µg/ml for single-stranded DNA.

# 4. Methods for determining DNA sequence

• Maxam-Gilbert method: Dividing DNA into segments according to each type of base (G, A, G + A, C, C + T).

• Sanger method: Using ddNTP (dideoxynucleotide) to stop DNA replication at specific locations.

# 5. DNA electrophoresis

• Type of gel: Agarose gel for horizontal electrophoresis and polyacrylamide gel for vertical electrophoresis.

• DNA migration direction: Inversely proportional to the size of the DNA molecule.

# 6. Conclusion

DNA extraction is a crucial technique in genetic research, molecular biology, and medicine. Understanding the steps and basic principles of the DNA extraction process is essential to ensure the quality and accuracy of research results.