ANDC DU/Biology Protocols/DNA Electrophoresis
Electrophoresis is the study of movement of charged particles in an electric field. Various types of electrophoresis are as follows:
Sample is applied on to the Whatmann filter paper or cellulose acetate paper moistened with the buffer solution. This method is used for separation of low molecular weight compounds such as amino acids, small peptides and nucleotides.
Starch gel electrophoresis
Starch gels are prepared by heating and then cooling a partially hydrolysed starch in an appropriate buffer. The separation is achieved due to the difference in charges as well as size. Isoenzymes can be separated by this method.
Polyacrylamide gel electrophoresis
It is used for the characterization and analysis of proteins ans nucleic acids. Polyacrylamide gel is prepared by polymerization of acrylamide monomers into long chains and cross-linking these bifunctional reagents with bis acrylamide. The pore size of polyacrylamide gels can be varied by changing total concentration of acrylamide and bis acrylamide solutions. The gel with low percentages has larger pore size and is suitable for separation of high molecular compounds and high percentage gel has smaller pore size and is used for separation of relatively low molecular weight compounds.
Agarose Gel Electrophoresis
Agarose gels are more porous and have a larager pore size as compared to polyacrylamide gels and are therefore used to fractionate large macromolecules such nucleic acids. Agarose is a linear polymer of d-galactose and 3,6-annhydro-L-galactose. Agarose gels are cast by boiling agarose in the presence of buffer, the poured into mold and allowed to harden to form matrix. Porosity is determined by the concentration of agarose. Higher the concentration, smaller the pore size and lower the agarose concentration larger the pore size. When electric current is applied across the gel, DNA molecules that are negatively charged at neutral pH migrates towards anode at rate determined by their molecular weights. DNA molecules of the same size but different conformations travel at different rates.
Any charged ion or molecule migrates when placed in an electric field. The rate of migration of a compound depends on its net charge, size, shape and the applied current. The movement of a charged molecule in an electric field is often expressed in terms of electrophoretic mobility µ which is defined as the velocity per unit of electric field. The electrophoretic mobility of a molecule is directly proportional to the charge density. Molecules with different charge/mass ratio migrate under the electric field at different rates and hence get separated. This is the underlying principle for all the electrophretic techniques.
The most convenient and commonly used method to visualise DNA in agarose gels is staining with fluorescent dye ethidium bromide which contains a tricyclic planar group which intercalates between the stacked bases of DNA. Ethidium bromide binds to DNA with little or no sequence preference. Ethidium bromide absorbs the UV radiation and energy is reemitted in red orange region of visible spectrum. Ethidium bromide can be used to detect both double and single stranded DNA.
Bromophenol Blue0.25 g
0.5M EDTA20 ml
Dissolve the sucrose in 50 ml of distilled water by heating and the add Bromophenol blue and EDTA. Raise the volume to 100ml.
TBE Buffer pH 8.3
Tris 108 g
Dissolve the components in 700 ml of distilled water and adjust the pH to 8.3. raise the volume to 1000 ml. sterilize by autoclaving.
Stock: 10 mg/ml in distilled water
Working: 0.5ug/ml in 0.5X TBE buffer.
- Horizontal electrophoresis apparatus
- Power pack
- Transilluminator/UV lamp
- Gel documentation system
- Make 1% agarose in 0.5X TBE buffer. Heat the mixture in boiling water bath or microwave until the solution becomes clear.
- The clean casting mold and place it horizontally on the table and level it using the equilibrium bubble. Position the comb0.5-1.0 mm above the plate so that a a complete well is formed when agarose is added.
- Pour the warm agarose solution in to the mold. There should not be any air bubble between or under the teeth of the comb.
- After the gel completely solidifies carefully remove the comb.
- Pour 0.5X TBE buffer over the cast gel
- Mix 15ul of DNA sample with 1.5ul of bromophenol blue tracking dye and slowly load the mixture into wells
- Connect the assembly with power supply and apply the voltage of 80 V.
- Run the gel until the bromophenol blue dye has traversed 3/4th distance.
- Turn off the electric current and remove the gel from the gel tank.
- Stain the gel with ethidium bromide for 20 to 30 minutes.
- Analyse the gel under UV light.
- Ethidium bromide should be handled carefully with gloved hands.
- Do not touch the gel when electric current is ON.
- Do not look at UV radiations with naked eyes.
|Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page.|