Gel Electrophoresis: DNA Fragment Separation

Study Notes

In gel electrophoresis, negatively charged particles move towards the anode, while positively charged particles move towards the cathode.

Gel electrophoresis separates DNA fragments by size in a solid support medium, such as agarose or polyacrylamide.
The process involves loading a DNA sample into sample wells, followed by the application of an electric current.
The negatively charged DNA then migrates towards the anode (positive) end.
The rate of migration is proportional to size, with smaller fragments moving more quickly and winding up at the bottom of the gel.

DNA is stained by including the fluorescent dye, ethidium bromide, in the gel or intercalating it.
As DNA fragments migrate through the gel, they take up the dye.
Ultraviolet light causes the intercalated dye to fluoresce.
The larger fragments fluoresce more intensely, although each fragment of a single class of molecule is present in equimolar proportions.

Agarose gel electrophoresis is a routine method for separating proteins, DNA, or RNA
Applications of agarose gel electrophoresis include:
- Estimating the size of DNA molecules
- Analyzing PCR products
- Separating restricted genomic DNA prior to Southern analysis
- Separating RNA prior to Northern analysis
- Estimating the size of DNA fragments after digesting with restriction enzymes

To prepare the gel, samples containing DNA are mixed with loading buffer and pipetted into the sample wells
The lid and power leads are placed on the apparatus
A current is applied, and a current flow can be confirmed by observing bubbles coming off the electrodes
During electrophoresis, DNA will migrate towards the positive electrode, usually colored red
The distance DNA has migrated in the gel can be judged by visually monitoring migration of tracking dyes