Gel Electrophoresis

Gel electrophoresis is a method used in laboratories to separate DNA fragments of different sizes. These sizes can then be estimated using a "DNA ladder" reference.

DNA, such as a PCR product, is loaded in small wells in one end of a gel block. An electric current is then applied to the gel (the positive electrode opposite to the wells). Because DNA is negatively charged, it will move through the gel toward the positive electrode. Smaller DNA molecules move faster through the gel than larger DNA molecules, leading to size separation. After electrophoresis, DNA is visualized and appears as bands under each well. Only a large amount of DNA (millions of copies)can be visualized. A DNA ladder containing a mixture of DNA fragments of known sizes is also loaded on the gel. The sizes of the DNA samples can then be estimated by comparing with the DNA ladder (Figure 1).

Figure 1. Fragments of different length from a PCR reaction are run on a gel. The fragments will move at a speed and a distance relative to their size: smaller DNA molecules will move down the gel faster and further than longer DNA molecules. Steps in gel electrophoresis. Two images presenting the gel electrophoresis apparatus, as vertically aligned blue rectangle with bands on top and agarose gel on the bottom, at time equal to 0 min and time equal to 45 min. Above the apparatus, where the samples are loaded, cathode is placed, and below the apparatus, anode is placed, and both of them are connected to the power supply. In the first step, DNA samples are loaded at the top of the rectangular apparatus. In the second step, an electrical field is applied. In the third step, DNA separation takes place, which can be seen on the second image, where small horizontal short bands are visible at different heights inside the blue rectangle. Fourth step is UV transillumination and documentation.

By mixing the PCR product with loading buffer, we can visualize how far the PCR product has traveled during gel electrophoresis. Loading buffer is also useful in making the sample heavier so that when you pipette it onto the gel, the sample will sink to the bottom of the gel.

There are many compositions for loading buffer; however, it will typically contain the following:

  • Coloring reagent, for example, xylene cyanol, cresol red, bromophenol blue, or orange G. This will add color to the sample and help you visualize the sample position on the agarose gel.

  • High-viscosity reagent, for example, Ficoll, sucrose, or glycerol. This will make the sample heavier by increasing the overall sample viscosity.

  • Water to dilute the above mentioned reagents.

PCR

Linkage analysis

Theory Overview