Gel electrophoresis helps in separating DNA fragments.
DNA fragments are negatively charged then they are forced to move towards anode under an electric field through agarose gel matrix. The fragments separate according to their size through sieving effect. Hence the smaller fragments move faster and further than the larger ones.
Separation and Isolation of DNA Fragments (Gel Electrophoresis)
▪ Gel electrophoresis is a technique for separating DNA fragments based on their size.
▪ Firstly, the sample DNA is cut into fragments by restriction endonucleases.
▪ The DNA fragments being negatively charged can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
▪ Commonly used matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-anhydro-L-galactose which is extracted from sea weeds.
▪ The DNA fragments separate-out (resolve) according to their size because of the sieving property of agarose gel. Hence, smaller the fragment size, the farther it will move.
A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments (lane 2 to 4)
▪ The separated DNA fragments are visualised after staining the DNA with ethidium bromide followed by exposure to UV radiation.
▪ The DNA fragments are seen as orange coloured bands.
▪ The separated bands of DNA are cut out and extracted from the gel piece. This step is called elution.
▪ The purified DNA fragments are used to form recombinant DNA which can be joined with cloning vectors.
