Pathognes Electroforesis

Pathogens Electrophoresis

Electrophoresis in 2% agarose gel „

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B.K.Kolita Kamal Jinadasa, Jinadasa, Post Harvest Technology Division, NARA, ColomboColombo-15, Sri Lanka.

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Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through through an agarose matrix with an electric field (electrophoresis). electrophoresis). Shorter molecules move faster and migrate further than longer ones. Components of electrophoresis in 2% agarose gel are: Agarose is purified from agar. agar. Different purities of agarose are commercially available as are agaroses with different melting properties. High purity low melt agarose is often used if the DNA is to be extracted from the gel. TBE or Tris/Borate/EDTA Tris/Borate/EDTA,, is a buffer solution that consists of a mixture of Tris base, base, boric acid, acid, EDTA, EDTA, and water. water. Ethidium bromide is an intercalating. intercalating. When exposed to ultraviolet light, light, it will fluoresce with a redred-orange color, intensifying almost 2020-fold after binding to DNA. DNA. Ethidium bromide is a very strong mutagen, mutagen, and may possibly be a carcinogen Color marker dye containing a low molecular weight dye such as "bromophenol blue" blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA solution more dense so it will sink sink into the wells of the gel

Preparation of electrophoresis. „

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Electroforesis

DNA marker consists of a plasmid that is digested to completion with appropriate restriction enzymes to yield DNA bands suitable for use as molecular weight standards for agarose gel electrophoresis A gel rack A "comb" Power Supply UV lamp or UV lightbox or other method to visualize DNA in the gel Preparation of a 2% agarose gel: Take 2 g of agarose and inoculate it into 100 ml of TBE. Heat the mix of agarosa and TBE until all agarose is diluted. Add 2.7 µl of Ethidium bromide. Shake the mix until all Ethidium bromide is diluted Empty the gel into the rack Put a comb in the gel Cool the gel about 10 minutes. Load the gel with samples + color marker dye Connect the gel to the power Watch the results with a UV lamp

Gel rack and combs

Load gel

Electrophoresis in 2% agarose gel

DNA marker

UV lamp

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Electrophoresis in 2% agarose gel „

Typical results

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