The goal of DNA electrophoresis is to separate DNA fragments by size on a porous gel matrix.
To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is added to the warm mixture, which is poured into the casting mold. A comb is inserted to form the wells.
Once solidified, the gel is transferred to a gel box filled with buffer, and the comb is removed. A reference mixture of dyed DNA fragments of known lengths—the DNA ladder—is added to one well, and the dyed DNA samples of interest are loaded into the remaining wells.
The box is connected to a power source, and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel, towards the anode—the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty.
When the run is complete, the gel is exposed to ultraviolet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands.
In the lab, first weigh out agarose, add buffer to create a percentage of solution that is suitable for the size range of DNA fragments, heat the mixture to dissolve the powder, and incorporate the nucleotide stain. After cooling, cast the mold and create the wells. Add loading dye to the samples to aid in gel loading and observing the migration process. Cover the surface with buffer, and carefully pipette DNA into the wells.
Run the gel by applying an electric field to separate pieces of DNA by size, and visually compare test samples to known bands of the ladder. The bands can be removed and combined with other purification and identification techniques.