Gel Electrophoresis:

                  ➤It is molecular technique which can separate samples like DNA, RNA or proteins based on their charge, length and size. This method is widely used in molecular biology, biochemistry, genetics and clinical biochemistry. 

              ➤Two different type of gels are used for separation of different types of samples, for the separation of DNA sample we use a separate gel called Agarose, when we are separating proteins from a mixture of proteins using a gel then we use a different gel known as polyacrylamide gel.

Principle: 

                Electrophoresis is a method of separating mixture of samples based on their size (molecular weight) and charge. The protein mixtures are separated based on their molecular weight and the DNA mixture sample is separated based on their length of DNA molecule, since all the DNA molecules are negatively charged. In protein there are both positively and negatively charged amino acids are present. In case of protein mixture separation first we need to unfold the protein, complex structure and impart negative on it. The gel matrix contains minute pores or mesh like structure through which the samples are passed when the electric field is applied. The samples moves from cathode (-) towards the anode (+).

Agarose gel electrophoresis: 

           ⮚For the separation of DNA from mixture of different length of DNA the agarose gel electrophoresis method is used. A gel matrix is created and the DNA samples or DNA fragments can be moved on the matrix based on the DNA length the movement varies. 

         ⮚The gel matrix is created by mixing the agarose powder in the buffer solution, after heated up to 40 degree Celsius and cool it down when we cool it down it form a semisolid gel like structure.

         ⮚Small wells are created while letting the solution to cool after the solidification the gel matrix is placed in the chamber which is connected to the electrode with different charges. The sample DNA fragments particles move from cathode (-) towards the anode (+). The chamber is filled with buffer solution to maintain the proper PH level.

         ⮚The DNA samples are loaded in the wells along with dye (to see the movement of fragments) and ladder (DNA fragment with known length to compare the samples), and the electric field id applied.

         ⮚While the gel is solidifying it forms a Mesh like structures and smaller pores are present in it. The smaller DNA can easily pass the pores then the larger DNA fragments during the electric field is applied. So, the larger fragments move slow and smaller moves fast as a result differential ability of their movement we can easily find out their differential position in the agarose gel.

         ⮚The size of the pores in the agarose gel matrix differ based on the concentration of agarose used. It differs based on their samples. When the dye reached the bottom line, the electric field applied is stopped.

         ⮚After that the gel matrix is taken out for staining. The DNA fragments are tagged with dye EtBr (Ethidium bromide) and then the gel matrix is placed under the UV for analysis. We can see different length of bands under UV thick band show larger DNA fragment and thin band is smaller size.

Materials used in agarose gel electrophoresis:

·        Electrophoresis chamber
·        Buffer solutions (tris – acetate -EDTA, glycerol)
·        Agarose gel
·        Gel casting tray
·        Comb
·        DNA Ladder
·        Staining agent
·        SAMPLES
·        Transilluminator

Application:

·        Used in northern and southern blotting for the DNA and RNA analysis

·        Analysis of PCR products

·        Helps to estimate the size of the DNA molecules

·        This method is widely used in molecular biology, biochemistry, genetics and clinical biochemistry.

References