Question

1. TAE buffer contains Tris base, acetic acid, and EDTA. Briefly describe what this buffer is used for and what are the specific functions of each component. Why is it important to use TAE buffer instead of water in situations in which TAE is recommended?

Answer 1

•Answer :- first we understand the meaning of Buffer solution

•A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it.

•Buffere is always added into the solution to keep the pH constant.

•Now let us focus on TAE buffer . What exactly it is :- TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

•Where it is used :- In molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as DNA and RNA.

•What is the pH of it :- It is made up of Tris-acetate buffer, usually at pH 8.3, and EDTA, which sequesters divalent cations.

•Applications

• Electrophoresis of nucleic acids in agarose and polyacrylamide gels
• Used both as a running buffer and as a gel preparation buffer
• Filtered through a 0.22 µm membrane
• Recommended for resolution of RNA and DNA fragments larger than 1500 bp, for genomic DNA and for large supercoiled DNA

•Role of Tris (hydroxymethyl) aminomethane (THAM) :- It is the main buffering component; its chief role is to maintain the pH of the buffer at a stable point, usually 8.0. Tris is an organic compound often used in buffer solutions such as TAE or TBE for electrophoresis gels. Tris is highly soluble in water and is useful in the pH range 7.0-9.0. It is used in the preparation of Laemmli buffer, one of the most common SDS-PAGE buffers.

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•Role of acetic acid :- Acetic acid is a weak acid because it doesn't dissociate very much in solution, meaning there are many more whole molecules of acetic acid than separate acetate and hydrogen ions.. In the buffer , the acetic acid hydrolyses and releases a hydrogen ion, which increases the acidity, to compensate.

•Role of EDTA :- The EDTA (ethylene-diamine-tetraacetic acid) molecule is a chelating agent widely used in molecular biology to sequester divalent and trivalent metal ions such as calcium and magnesium. This ability prevents DNA and RNA degradation as metal-dependent enzymes acting as nucleases becomes deactivated. EDTA is tetravalent and has four pK values. Hence it conducts more electric current producing more heat.

•A buffer is used in gel electrophoresis instead of water because it helps maintain the pH. During gel electrophoresis, samples are loaded into small holes called wells at the top of the gel.