The principle that allows electrophoresis to separate different DNA fragments, despite all fragments having the same charge, is based on their size. When an electric field is applied during electrophoresis, DNA fragments will move towards the positive electrode due to their negative charge (from the phosphate backbone).
However, the gel matrix (typically made of agarose or polyacrylamide) acts as a mesh-like structure that hinders the movement of larger DNA fragments more than smaller ones. Smaller fragments can navigate through the pores of the gel more easily and therefore move faster through the gel matrix when subjected to the electric field. Conversely, larger fragments experience more resistance and migrate more slowly.
As a result, when the electrophoresis is complete, DNA fragments are separated based on their size—the smaller fragments will be positioned further away from the wells (where the DNA was loaded) compared to the larger fragments. This size-based separation allows for the analysis of different DNA fragments effectively.
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