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Here, I have described different types of commercially available gel extraction kit for the gel excision. These are listed below in a table summarizing the most important comparative information about different kits. These commercially available gel extraction kit can easily be utilized for the DNA gel excision.
The basic principle is that the DNA fragment selectively binds to the spin column with built-in silica membrane. Then centrifugal force allows the passage of solutions and other contaminants while retaining the DNA into the spin column. The spin column-bound DNA can be eluted with an elution buffer. Each gel extraction kits have their own protocol and components but their basic principles are same.
Dissolve the piece of gel containing DNA in an appropriate binding buffer in a ratio of 1:3 or 1:4. Incubate the gel-binding buffer mixture in a water bath of 50- 60 °C for 10 minutes (Note-1). Load the mixture into the spin column with built-in silica membrane followed by centrifugation. Please follow the manual provided in the kit box. During the centrifugation, DNA binds to the silica membrane and rest of the components pass through the membrane.
Wash the spin column-bound DNA with ethanol containing wash buffer for up to two times. Washing further removes contaminants such as nucleotides, salts and other impurities. After that, centrifuge the spin column with no wash buffer to ensure that the spin column is free of ethanol.
Finally, elute the DNA bound to the spin column in a clean Eppendorf tube using pre-warmed elution buffer (Note-2). Thus eluted DNA sample is pure and ready to use in downstream applications or for sequencing.
Note-1: The binding buffer contains chaotropic agents and a color indicator to make sure that the mixture has an optimal pH for DNA binding. Chaotropic agents such as guanidinium chloride break the H-bonding between the agarose and water molecules. The yellow color of the binding buffer indicates the optimal pH for DNA binding.
If the color of the gel and binding buffer mixture is orange or violet, then add 10 µl of 3 M sodium acetate of pH 5.2 solution. This is to make sure that the mixture has an optimal pH for DNA binding as indicated by the yellow color.
Note-2: If the DNA fragment is higher than 10 kb, you need to pre-warm the elution buffer to 65 °C before eluting the column-bound DNA.