Describe, in detail, to a freshman undergraduate student the basic principle underlying the separation of dna using agarose gel electrophoresis. explain the difference between a running buffer and a loading dye.

Definition

A technique which is used to separate, DNA, RNA or protein pieces from each other under the influence of electric field on the basis of their molecular size is known as gel electrophoresis.

Explanation

This method is very reliable for separation of large size molecule (over 1 million Da). Materials which are required for gel electrophoresis include:

1. TAE stock buffer

2. 1% agrose gel

3. Nucleic acid loading dye

4. Ethidium bromide

Procedure:

First prepare a stock solution of TAE buffer by adding appropriate amount of TAE in distilled water. then prepare 1% agrose by adding 1X TAE, some amount of agrose in water and heating it in microwve oven to mix them will. then pour agrose gel on tray and fix comb in it and keep it untill agrose dry. Then remove comb and pour some quantity of nucleic acid along with loading dye and ethidium bromide in each well. EtBr is used for staining nucleic acid.

When sample is poured in all well also pour reference marker in one well for comparison. now connect it with voltage for 30-35 min. After this take gel and see it under UV. a large number of nucleic acid pieces will be seen on gel under UV. those pieces which have small molecular weight will cover more distance compared to those having larger molecular weight.

Electrophoresis is a laboratory technique performed with the objective of separating molecules according to their size and electrical charge so that diseases can be diagnosed, protein expression verified or microorganisms can be identified.

The difference between a buffer and a loading dye is that the buffer is used to increase the sample density and color the sample, while the loading dye is used to color the sample and allow visualization of the electrophoresis result. under ultraviolet light.

Explanation:

Electrophoresis requires the gel, which may be polyacrylamide or agarose depending on the purpose, electrophoresis buffer and vat, molecular weight marker and fluorescent dye, as well as UV or LED light equipment, also known as a transilluminator.

After preparation of the gel, a specific object should be placed to make the wells in the gel, popularly called comb, and let the gel settle. When the gel is ready, simply apply the substances to the wells. For this, a molecular weight marker must be placed in one of the wells, a positive control, which is the known substance, a negative control, which guarantees the validity of the reaction, and the samples to be analyzed. All samples should be mixed with a fluorescent dye, as this allows visualization of the bands on the transilluminator.

The sample gel should be placed in the electrophoresis vat, which contains the specific buffer solution, and then the device is turned on so that there is electric current and therefore potential difference, which is important for separation. particles according to their charge and size. The electrophoretic run time varies according to the purpose of the procedure and may last up to 1 hour.

After the given time, it is possible to view the result of the electrophoretic run through the transilluminator. When the gel is placed under UV or LED light, the band pattern can be seen: the larger the molecule, the smaller its migration, getting closer to the well, while the lighter the molecule, the greater the migratory potential.