Lysis buffers

You: The lysis buffer is responsible for denaturing proteins and helping in the breakdown of tissues and cells.

Dr. Demetriou: Well-done! This is correct. Do you remember what it contains to do this?

You: Doesn’t the lysis buffer contain detergents? 

Dr. Demetriou: That’s right, the detergents help to break open cells and tissues, releasing their contents, including DNA. It also helps to denature proteins, making them more accessible to digestion by Proteinase K. Remember that in the first part of the DNA isolation process, proteinase K is added to the lysis buffer (ATL). 

[The Proteinase K enzyme digests proteins, including nucleases that could degrade DNA, and helps to lyse cells and tissues. It ensures that the DNA is released from protein-DNA complexes and remains intact during the purification process.]

You: This was very helpful Dr. Demetriou. By understanding the purpose of each reagent, I can now better appreciate how the DNA purification process works. I understand that high-quality DNA is needed for the PCR. I will go ahead and isolate DNA from mouse tails. 

Dr. Demetriou: Best of luck with your first DNA isolation. 

[Buffer AL/Ethanol: Buffer AL contains chaotropic salts, which help to lyse cells and denature proteins and nucleic acids. It also aids in the binding of DNA to the silica membrane in the spin column by disrupting hydrogen bonds and hydrophobic interactions. Ethanol is used to precipitate DNA and enhance its binding to the silica membrane in the spin column. It helps to remove contaminants and facilitates the washing steps by making the DNA less soluble in the aqueous phase.]

Wash buffer 1 (AW1): The column containing the sample is washed with Buffer AW1. This wash buffer contains a low concentration of chaotropic salts and ethanol. It helps to remove proteins, salts, and other contaminants from the DNA bound to the silica membrane without eluting the DNA.

Wash Buffer 2 (AW2): The column containing the sample is washed with Buffer AW2. This wash buffer contains ethanol but no chaotropic salts. It further cleans the DNA by removing any remaining contaminants, such as salts and residual proteins, ensuring that the final DNA preparation is pure.

Buffer AE (or distilled water): This elution buffer is used to release the purified DNA from the silica membrane. Buffer AE typically contains Tris-EDTA, which helps to stabilize the DNA and protect it from degradation. Distilled water can also be used if a more neutral elution is desired.