Question

Which of the following are the commonly used solvents employed to dissolve agarose while gel preparation?
I. TAE
II. TBE
III. Water
IV. Oil

• A. I and II
• B. I, II and III
• C. I, III and IV
• D. I, II, III and IV

Answer 1

The correct option is A I and II

Agarose gel electrophoresis is a technique commonly utilised for the separation of a mixture of DNA (a negatively charged molecule) fragments of varying sizes on agarose gel (a natural biopolymer) matrix under the influence of electric field. The solidified gel has pores of certain sizes that provide a sieving effect to separate the DNA fragments of different sizes.

During gel preparation, the agarose powder is accurately weighed (in order to get the gel with desired pore sizes). This is then dissolved in a solvent to achieve a particular concentration. The solvent used is a buffer. A buffer is a solution that resists a change in its pH when a small amount of acid or base is added externally. The commonly used buffers in this case are TAE and TBE.

• Tris helps to maintain constant pH of the solution.
• EDTA helps to chelate the divalent ions like Mg${}^2$${}^{+}$, which if present can activate nucleases and hence, degrade our samples.
• Acetic acid and boric acid provide the appropriate ion concentration in the buffer, that is, in TAE and TBE respectively. These ions help in conduction of electric current that help in the migration of sample DNA.

TAE favors high conductivity while TBE supports better cross linkage in agarose gel.

After dissolution of agarose in the buffer, it is heated to get a homogenous mixture. This is then casted and allowed to cool to get the gel.

Water is not used as a solvent for agarose as it lacks buffering action and also contains ions like Mg${}^2$${}^{+}$ which can activate nucleases which may destroy the DNA sample.

Oil is an organic liquid, non-polar in nature. The DNA molecule, being negatively charged and polar, would not be able to pass through it. Besides, oil lacks any buffering capacity or ions, hence would not be able to conduct electricity.

Hence, water and oil cannot be used as solvents for dissolving agarose to prepare gel for electrophoresis.