Agarose gel electrophoresis is a widely used method to separates molecules based upon charge, size and shape. It is particularly useful in the separation of charged biomolecules such as DNA, RNA and proteins.
This course covers agarose, advantages of agarose gels, general principle involved in agarose gel electrophoresis, and its applications.
Advantages of Agarose Gel
Agarose gels are convenient to separate DNA from few hundred base pairs or more. There are several advantages listed below in using agarose:
- Agarose is non-toxic and unlike polyacrylamide, contains no potentially damaging polymerization by products.
- Agarose allows rapid diffusion of high molecular weight (106 dalton range) macromolecules without significant restriction by the gel.
- Rapid staining and destaining can be performed during the procedure with minimal background.
- Agarose gels are thermoreversible and can be air dried.
Factors such as charge, size and shape, together with buffer conditions, gel concentrations and voltage, affect the mobility of molecules in gels.
If there are two molecules of the same molecular weight and shape, the one with the greater amount of charge migrates faster. This phenomenon is called the sieving.
Nucleic acids get separated according to their sizes, whereas proteins are separated according to their charges in agarose. The pores in the gel are too large to sieve protein molecules based on their size.
- A 10-30 μl/sample of the DNA fragments to be separate and mixture of DNA fragments (usually 10-20) of known size (after processing with DNA size markers either from a commercial source or prepared manually).
- Buffer solution usually TBE buffer or TAE 1.0x, pH 8.0
- An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O) An alternative dyes such as SYBR Green (cyanine dye) can be used
- A color marker dye such as bromophenol blue and glycerol
- A gel rack, a comb, power supply and UV lamp or UV trans-illuminator to visualize DNA in the gel (Figure 3)
When the dye approaches the end of the gel, stop the current. Stained the DNA fragments with ethidium bromide, and observe the bands under UV trans-illuminator.
- Prepare 50X TAE
- 242 g Tris Base
- 57.1 ml Glacial Acetic Acid
- 100 ml 500 mM EDTA, pH 8.0
- 600 ml ddH2O
Mix above solution and bring the volume to 1L and autoclave.
- Mix the following
- 0.40 g Agarose
- 4.00 ml 50X TBE (4X TAE final)
- 46.00 ml Water
- 1.00 µl 10mg/ml Ethidium bromide
- Melt agarose in a microwave.
- Seal horizontal gel apparatus. Pour molten agarose onto gel plate to a depth of 4-8mm.
- Insert a comb until its base is 1mm from the base of the gel. Allow it to cool.
- Remove comb and submerge in 4X TAE buffer.
- Prepare 6X loading dye as
- 3 ml 100% Glycerol
- 3 ml 0.5 M EDTA, pH 8.0
- 3 mg Bromophenol blue
- 3 mg Xylene Cyanol
- 4 ml Sterile Water
- 10 ml Total Volume
- Add 1/5 volumes 6X loading dye to sample. Mix well and pellet in a microfuge.
- Add sample to well with a pipette. Do not overflow well. For large samples, either reduce sample in volume prior to adding dye, or load into multiple wells.
- Continue until bromophenol blue comes at the end of the gel.
- Remove gel and visualize bands under UV light (Figure 4).
Applications of Agarose Gel Electrophoresis
- It is used to type DNA to study the heritable diseases.
- Mapping traits in segregating populations can be assessed using this technique.
- Identification of diseased genes including oncogenes, viral infections, and tissues found at a crime scene.
- DNA profiling done by gel electrophoresis that determines genetic similarity and kinship among population.
- SDS-PAGE is most commonly used for protein analysis.
- 2-D PAGE is used for the determination of purity of the protein samples.