Agarose Gel Electrophoresis of DNA

DNA agarose gel electrophoresis is an analytical technique of analyzing and separating DNA fragments based on their size. Agarose gel electrophoresis utilizes agarose gel, a porous matrix,  to separate electrically charged molecules under an electric field. Since DNA is negatively charged (due to phosphate group) at neutral or slightly basic pH, it moves under an electric field from the cathode (negative electrode) to the anode (positive electrode) through the gel matrix which retards the movement of DNA fragments based on their size (larger size DNA fragments are retarded higher extent than small fragments), thus can easily be separated on an agarose gel. 

Unlike RNA, linear double-standard DNA fragments are usually separated on non-denaturing agarose gels. Agarose gel is prepared using a casting module, the casting tray, and comb (to create wells in the agarose gel for sample loading). Gel is allowed to solidify that takes ≈ 30 – 45 min. 

The solidified gel is then placed in an electrophoretic apparatus in horizontal position. The electrophoretic apparatus is equipped with two electrodes: cathode at one end and anode at another end. Electrophoretic buffer is then filled in the chamber to submerge the gel (horizontal agarose gel electrophoresis). 

Samples premixed with DNA loading dye are loaded onto the wells of the gel and electrophoresed under specified electrophoretic conditions. After the electrophoresis is over, gel is analyzed for DNA fragments. To visualize the DNA fragments in agarose gel, DNA staining dyes such as ethidium bromide, Gel Red etc can be used. DNA staining dye can either be added in the gel while casting or the gel is stained after the electrophoresis.

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