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In this article we will discuss about the principle, requirements and procedure for Agarose gel electrophoresis.
Agarose gel electrophoresis is a simple and highly effective method for separating, identifying and purifying DNA fragments. Separation is carried out under an electric field applied to gel matrix. Because of the negatively charged phosphates along the backbone, DNA molecules migrate towards the anode.
The rate of migration of a particular linear DNA fragment is inversely proportional to the ratio of its molecular weight. Separation of DNA molecules is strictly based on size, the smallest fragments move further in the gel, since they can navigate through the small pores in the gel, compared to larger molecules.
There are various parameters like concentration of agarose, voltage, electro-phoresis buffer, and DNA concentration that affect the migration of DNA through agarose gel and hence critical selection should be made of these parameters.
DNA molecules between the size 500— 2500 bp can be separated and identified. Subsequently from a heterogeneous mixture, purification of DNA molecules of interest can be done. On a gel, if there are a series of size standards, the actual size of a particular size of DNA can be determined from its distance of migration. This method is useful in gene manipulation experiments.
Principle:
Agarose, a copolymer of D-galactose and 3, 6 anhydro L-galactose, forms a gel by hydrogen bonding. Concentration of agarose determines its pore size. The resolution of larger fragments of DNA are better with low concentrations of agarose, whereas it reduces the resolution of smaller fragments.
Agarose gel electrophoresis is conducted in a horizontal configuration because it provides better support especially at low agarose concentration and the bands of DNA are less distorted and the gel should be completely submerged in the electrophoresis buffer.
Requirements:
Reagents:
Materials:
Horizontal gel electrophoresis apparatus. Gel casting platform, gel combs (slot formers)
1. DC power supply unit.
2. Microwave oven.
3. U.V. transilluminator.
4. Micropipette.
5. Parafilm.
6. Adhesive tapes.
7. Eppendorf tubes, tips.
8. Gel scoop and conical flask.
Procedure:
1. Seal the ends of the plastic gel casting platform with an adhesive tape.
2. Prepare 0.8% agarose by mixing in lx electrophoresis buffer, boil in a microwave oven with intermittent stirring till a homogenous dear solution is formed and cool to 55°C.
3. Add ethidium bromide to a final concentration of 0.5 µg/ml (5 µl of 10 mg/ml solution of ethidium bromide solution to 100 ml gel mixture) and mix well.
4. Pour the molten gel into the gel mould, place the comb and allow it to set for atleast 30 minutes (while placing the comb, its teeth should be away from the bottom of the plate, atleast 1 mm) on a horizontal leveling-table.
5. Remove the comb carefully and pull the tape off the gel casting platform. (Selection of gel casting platform, percentage of gel, thickness of gel and dimensions of electrophoresis unit vary as per the quantity and nature of the samples to be electrophoresed).
Care should be taken to see that there is no air bubbles trapped while pouring the gel since it affects the migration of DNA fragments during electrophoresis and transfer of DNA during southern blotting. The gel must be cold while removing the comb; otherwise the DNA fragments are likely to ‘smile’ when they are run on the gel. Care should also be taken to see that the wells are not disturbed while removing the comb.
6. Keep the gel in an electrophoresis unit with lx electrophoresis buffer. The gel should be completely immersed in the buffer.
7. Attach the electrodes making sure the negative (black) terminal is at the same end of the unit as the wells.
8. As a pre-run, run at 20 mA power.
9. Add 1/10 volume of tracking dye (10x stock) to the DNA sample and mix by tapping. Spin in a microfuge for 30 seconds.
10. Turn off the power supply and load the DNA sample carefully. Load a suitable molecular weight marker in the first as well as the last lane along with DNA samples. Turn the power supply on and adjust current to 50 mA (while loading, care should be taken to see that the tips should not touch the front and back walls of the well, since this will result in a streaky lane) using 1x (TAE) buffer for running.
11. Check that the DNA samples are migrating in the right direction to the anode, (black to red). Over-heating of gel and distortion of bands will occur if higher currents are used.
12. Turn off the power supply. Bromophenol blue co-migrates with 500 bp fragments.
Gel staining and viewing
13. Remove the gel from electrophoresis unit and immerse it in ethidium bromide stain (10 mg/ml) for 20-30 minutes. (An alternative is to add ethidium bromide to the agarose gel and running buffer and viewed on a transilluminator directly). Ethidium bromide is a powerful mutagen and hence gloves should be worn while handling it and separate trays and glasswares should be used.
14. View the gel on U.V. transilluminator (with protective eyeware) and photograph the gel using gel documentation and store it in computer.