Gel electrophoresis is a laboratory technique used in genetics to separate mixtures containing DNA, RNA, and other proteins according to their respective molecular size and charge. The DNA, RNA, or proteins that must be separated in this method are run through a gel that contains small pores. The molecules are driven through the gel by an electric field.
Horizontal gel electrophoresis
Horizontal gel electrophoresis uses the basic theory for the separation of DNA, RNA or protein molecules according to their respective molecular size and charge. In this technique, the gel is present in a horizontal orientation and is immersed in a buffer that is continuous. The agarose gel is used to separate the gel box into two compartments. One end of the gel box contains an anode, while the other end contains a cathode. When a current is applied, the buffer used in this technique allows the creation of a charge gradient. When the load is applied, the gel tends to heat up.
The buffer also functions as a coolant, keeping the temperature at optimal levels. Recirculation of the running buffer prevents the formation of a pH gradient. A discontinuous buffer system cannot be used in horizontal gel electrophoresis as the two compartments of the gel system connect with the running buffer.
In horizontal gel electrophoresis, acrylamide cannot be used as the gel box is exposed to oxygen. Due to the presence of oxygen, acrylamide polymerization is inhibited, which interferes with gel formation. Horizontal gel electrophoresis is an effortless method used in the separation of DNA and RNA.
Vertical gel electrophoresis
The vertical gel electrophoresis technique works according to the primary theory of gel electrophoresis, but is considered to be more complex than the horizontal gel electrophoresis method. This technique uses a discontinuous buffer. A cathode is located in the upper chamber, and the anode is located in the lower chamber. The electrodes present in each compartment provide the required electric field. A thin layer of gel is poured between the two mounted glass plates. Therefore, the upper part of the gel is immersed in the upper chamber, and the lower part of the gel is immersed in the lower chamber. Once the current is applied, a small portion of the buffer moves to the lower chamber from the upper chamber through the gel.
In vertical gel electrophoresis, the buffer only flows through the gel. This allows precise control of the voltage gradient during the separation stage. Acrylamide gel can be used as the compartments are not exposed to atmospheric oxygen. Due to the smaller pore size of the acrylamide gel, precise separation can be achieved with higher resolution.
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