IGEM:Hong Kong HKUST/Investigations/Effect of agarose concentrations on separation of linear DNA in gel electrophoresis
Investigation of the effect of agarose gel concentration on the separation of different size of linear DNA to find the best gel concentration for separation of certain range of DNA size.
Electrophoresis is a method to separate substances based on the rate of movement under an electric field. Agarose is a polysaccharide purified from seaweed. The DNA is pulled through the pores in the gel by electrical current. DNA will move toward the positive electrode and away from the negative electrode. There are several factors affecting the speed of DNA movement during electrophoresis such as: strength of electrical field, concentration of agarose gel and size of the DNA molecules. Smaller DNA molecules move faster than the larger ones. DNA itself is not visible within the gel and should be visualized using a dye that binds to DNA.
With constant voltage and running time, separation of longer DNA will be clearer with a lower agarose gel concentration whereas separation of shorter DNA will be clearer with a higher agarose gel concentration.
Instead of using other DNA, we used commercial DNA ladder. DNA ladder has more different sizes of linear DNA than self-cut extracted linear DNA.
The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. As the total amount of agarose powder increases, the pore size decreases. The pore size of the gel decreases due to increased rate of nucleation and closer packing of the chain. Shorter DNA, which is 100 bp, moves faster and migrates farther than longer ones in higher concentration because shorter DNA migrates more easily through the decreasing pore size of the gel. According to the result photo, 100 bp DNA ladder is best observed with 2.0% concentration as the bands are separated clearly, while in other gel concentration, the 100 bp DNA ladder is stacked together. Longer DNA, which is 1 kb, moves faster and migrates farther than shorter ones in lower concentration because longer DNA migrates more easily through the increasing pore size of the gel. According to the result photo, 1 kb DNA ladder is best observed with 0.5% concentration as the bands are separated clearly, while in other gel concentration, the 1 kb DNA ladder stacked together and become smear. This phenomenon is called sieving.
1. Measure 0.1 gram agarose powder using the electronic balance for 0.5% gel concentration