Question

What buffers are recommended for electrophoresis of DNA?

Answer 1

Electrophoresis:

1. Electrophoresis is defined as the laboratory technique used to separate DNA, RNA, or protein molecules on the basis of their charge and the size of the molecules.
2. Electric current is applied to move the molecules across the gel or the matrix.

Gel electrophoresis:

1. The DNA is isolated and the solution is prepared by adding blue dye.
2. An agarose TAE gel solution is prepared.
3. The agarose TAE solution is then poured into a casting tray, and allowed to stand cool and solidify.
4. The gel is placed in the chamber for electrophoresis by positioning the desired sample near the cathode rod.
5. Electrophoresis is carried out for some time.
6. The migration of blue colored DNA samples is observed and the power supply is switched off.
7. The gel is removed and placed in the ethidium bromide solution.
8. The gel stained with ethidium bromide solution stained is kept under UV light and is observed.

Buffers:

1. Buffers are said to be a mixture of weak acid and its conjugate base as well as vice versa.
2. The recommended buffers are:

1. Tris acetate EDTA (TAE) and tris borate EDTA (TBE) are two most common running buffers used in nucleic acid electrophoresis.
2. As buffers, they also have a fairly constant pH & are able to conduct electricity because of their concentration of hydrogen ions.
3. Agarose gel electrophoresis of the DNA and RNA is routinely performed using the buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE).
4. TAE also works better for cloning, because TBE contains borate. Borate in TBE is an inhibitor for many kind of enzymes, such as ligase. TAE works better for performing the DNA extraction from agarose gel.