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Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Samples are prepared for electrophoresis by mixing them with loading dyes. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA). The volume of the buffer should not be greater than 1/3 of the electrophoresis chamber. A solidified agarose gel after removal of the comb (Image Source: Ref-1)Īfter the gel solidifies, the gel is submerged in a buffer-filled electrophoresis chamber which contains a positive electrode (anode) at one end, and a negative electrode (cathode) at the other. A well-former template (often called a comb) is placed across the end of the casting tray to form wells when the gel solution solidifies. The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The solution is then cooled to approximately 55☌ and poured into a casting tray which serves as a mold. The centerpiece of agarose gel electrophoresis is the horizontal gel electrophoresis apparatus. The gel is made by dissolving agarose powder in boiling buffer solution. Procedure Preparation of Agarose gel matrix Within a range, the higher the applied voltage, the faster the samples migrate. Mobility of DNA molecule is also affected by the applied voltage. This means that the smaller the linear fragment, the faster it migrates through the gel. The migration rate of linear fragments of DNA is inversely proportional to the log 10 of their size in base pairs. plasmid DNA) moves faster through gel compared with linear DNA fragment of the same size. DNA conformationĭifferent forms of DNA move through the gel at different rates DNA molecules having a more compact shape (e.g. If the size of the two fragments is similar or identical, they will migrate together in the gel. The separation occurs because smaller molecules pass through the pores of the gel more easily than larger ones. The sieving properties of the agarose gel influence the rate at which a molecule migrates. However, some experiments require agarose gels with a higher percentage, such as 1% or 1.5%. The most common agarose gel concentration for separating dyes or DNA fragments is 0.8%. Higher percentage gels are sturdier and easier to handle but the mobility of molecules and staining will take longer because of the tighter matrix of the gel. The mobility of DNA molecule is inversely proportional to gel concentration.

1.0.5 DNA concentrations can be estimated by: The rate of migration of a DNA molecule through a gel is determined by the following parameters: Agarose concentration:.

1.0.3 Applying electric current and separating biomolecules.

1.0.1 Preparation of Agarose gel matrix.

0.1 The rate of migration of a DNA molecule through a gel is determined by the following parameters:.

Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. Agarose gel electrophoresis experiment overview (Image Source: Ref-2) In general, the higher the concentration of agarose, the smaller the pore size. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. The separation medium is a gel made from agarose. Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis whereas pulse-field gel electrophoresis is used to separate DNA fragments larger than 25 kb. The agarose gel consists of microscopic pores that act as a molecular sieve that separates molecules based upon the charge, size and shape.Īgarose gel electrophoresis is a powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes, and it is a convenient analytical method for separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Agarose gel electrophoresis is one of the most common electrophoresis techniques which is relatively simple and straightforward to perform but possesses great resolving power.