Qualitative Analysis of DNA
separate and visualize DNA bands by Agarose gel electrophoresis
Aim: To separate and visualize DNA bands by Agarose gel electrophoresis
Agarose gel electrophoresis is a powerful and widely used method that separates molecules on the basis of the electrical charge, size, and shape. The method is particularly useful in separating charged biologically important molecules such as DNA (deoxyribonucleic acids), RNA (ribonucleic acids), and proteins. Agarose forms a gel-like consistency when boiled and cooled in a suitable buffer.
The agarose gel contains molecule-sized pores, acting like molecular sieves. The pores in the gel control the speed that molecules can move. DNA molecules possess a negative charge in their backbone structure due to the presence of PO4- groups thus this principle is exploited for its separation. Smaller molecules move through the pores more easily than larger ones. Conditions of charge, size, and shape interact with one another depending on the structure and composition of the molecules, buffer conditions, gel thickness, and voltage. Agarose gels are made with between 0.7% (provides good resolution of large 5–10 kb DNA fragments) and 2% (good resolution for small 0.2–1 kb fragments). The gel setup provides wells for loading DNA in to it. The loaded DNA molecules move towards the positively charged electrode (anode) and get separated along the length of the gel. Ethidium bromide (EtBr), a chromogen is added to the gel to visualize the separated DNA under UV transillumination. EtBr intercalates between the bases and glows when UV radiation is passed through the gel
Purpose of gel loading buffer
The loading buffer gives color and density to the sample to make it easy to load into the wells. Also, the dyes are negatively charged in neutral buffers and thus move in the same direction as the DNA during electrophoresis. This allows you to monitor the progress of the gel. The gel loading dye possesses bromophenol blue and xylene cyanol. Density is provided by glycerol or sucrose.
Xylene xyanol gives a greenish blue color while bromophenol blue provides a bluish colored zone. The successful DNA run is determined by the presence of both the colored dye in the gel
• Electrophoresis buffer: 1x TAE buffer
• Agarose ultrapure (DNA graded)
• electrophoresis tank, gel tray, sample comb, and power supply
• Plastic or insulation tape
• Ethidium bromide: 10 mg /ml stock solution
• 5x Gel loading dye
• DNA marker solution, DNA sample, and gloves
1. Making a 1% Agarose Gel
• Weigh 0.5 g agarose and dissolve it in 50 mL of 1x TAE Buffer. (Note: Use 250 ml conical flask for preparing 50 ml solution to avoid overflow of gel solution while heating and to avoid its loss.)
• Heat the solution over a hot plate to boiling constituency marked with a clear solution
• Leave the solution to cool and add 2μl of EtBr solution mix it well by gentle swirling.
• Pour it in the gel tray-comb set up. Also be sure the gel plates have been taped securely and contain the well combs prior to pouring
• Allow the solution to cool and harden to form gel
2. Loading of Samples
• Carefully transfer the gel to the electrophoresis tank filled with 1x TAE buffer. • Prepare your samples [8 ul of DNA sample (0. 1 ug to 1 ug) and 2 ul of 5x gel loading dye]
• Remove the comb and load the samples into the well.
• Connect appropriate electrodes to the power pack and run it at 50100volts for 20min.
• Monitor the progress of the gel with reference to tracking dye (Bromophenol blue). Stop the run when the marker has run 3/4 th of the gel. 3. Examining the gel
• Place the gel on the UV-transilluminator and check for orange colored bands in the gel
- Wear gloves during the addition of EtBr and while handling the casted gel (EtBr is a potent carcinogen).
- Handling the gel should be careful as the gel may break due to improper handling.
- While performing the UV-trans illumination for visualizing the bands, avoid direct contact and exposure to eyes