Agarose gel electrophoresis is the most common way of separating and analyzing DNA.An agarose is a polysaccharide polymer material, generally extracted from seaweed. Agarose is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is available as a white powder which dissolves in near-boiling water, and forms a gel when it cools. The melting and gelling temperature may be dependent on the concentration of the gel. It is more solid and less elastic compared to a polyacrylamide gel.
The main purpose of using a Agarose gel is to separate nucleic acids(mostly DNA) electrophoretically because its gels have larger pore sizes than polyacrylamide gels at low concentrations. The separated DNA bands can be used for isolating or eluting a particular band.The DNA is visualized in the gel by addition of ethidium bromide, GelRed, GelGreen, and SYBR Safe. Ethidium bromide and other dyes bind to DNA and are fluorescent, meaning that they absorb invisible UV light and transmit the energy as visible light. Ethidium bromide is much more toxic compared to other dyes but still widely used as it is less expensive and gives strong signal.
Preparing 2% Agarose gel:
Gels are prepared in terms of percents: say 0.7%, 0.8%, 1%, or 2% are pretty common gel percentages. The percentage of the gel you use depends on the fragment size you’re expecting and how good you need to separate the fragments, and whether or not you will use DNA extracted from slices of gel for cloining or other purposes, after you electrophorese them. A 0.7% gel is generally used for resolving large DNA fragments (5–10 kb) and a 2% gel will show good resolution for small fragments (0.2–1 kb). One has to increase the percentage of Agarose to 3% for even smaller fragments a vertical polyacrylamide gel is considered a much better option.
The percentage measurement is a weight/volume thing. For example if you want to make a 100% gel, one should weigh 100g agarose in 100mL TAE or TBE. (TAE is Tris-Acetate-EDTA and TBE is Tris-Borate-EDTA); these are commonly used buffers for making and running gels for electrophoresis.) You wouldn’t make a 100% gel, though, that was just an example. More commonly, a 1% gel would be used and for that 1g agarose should be dissolved in 100mL TAE.
As we are interested in making 2% gel : we need to take weigh 2grams of agarose and dissolve it into 100ml of buffer or 0.5g/500mg in 50ml of buffer. In similar way you need to weigh 0.7g or 700mg in 100ml and .35g /350mg in 50ml of buffer to make 0.7% gel. These contents are heated in a microwave till Agarose is completely dissolved ( time depends on amount of Agarose used). Let it cool on the benchtop for five or so minutes (if you’re in a big rush you can run water over the surface of the flask to cool it down). Next add ethidium bromide or any non toxic dye: These are chemicals which intercalates with DNA and makes it visible under UV light.