Question

What is the purpose of 1% agarose gel electrophoresis? Why is TAE buffer used during this procedure?

Answer 1

Agarose gel Electrophoresis is very important tool in molecular biology for separation of biomolecule like DNA and protein. In these techniques DNA seperate from mixed solution in a matrix of Agarose gel. Agarose form a 3d matrix with have larger pore size. In the laboratory 0.7% to 2% agarose used. High concentration of agarose used when DNA fragments in the sample is very small in size. But one major problem is high percentage agarose matrix is very weak and easily break. So, used 1% as standard agarose concentration with 100-500 nm pore size. And almost all types of DNA fragments seperate easily. But if very high DNA fragments Sepetaration required then used very low concentration Agarose.

TAE buffer have pH 8-8.5, it is used in agarose gel electrophoresis for proper Sepetaration of DNA molecule TAE contain:- Tris base, Acetic acid, and EDTA.

TAE buffer act as a alkaline buffer during electrophoresis, EDTA act as a chelating agent, that binds Mg2+ ion. DNAase enzyme required Mg2+ for enzymatic activity, but EDTA preventing this activity by sequestered this ion. And helps DNA remains intact in gel.

During Electrolysis H+ ion also generate from water. And This positive ion may binds to negative backbone of DNA(phosphate group) and neutralized DNA charge, results in improper DNA Sepetaration. To prevent this problem Tris acetate act as buffer, menas it bind all H+ maintain constant pH.