Gel electrophoresis is a method used in laboratories to separate DNA fragments of different sizes and to estimate these sizes. DNA, for instance, a PCR product, is loaded in small wells in one end of a gel. 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).
图1所示。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.
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.
- 水to dilute the abovementioned reagents.
Plasmid
Uncut plasmid have five conformation, which is nicked, linear, covalently bound, supercoiled, and circular single stranded. A supercoiled plasmid travels faster because it has less friction against agarose matrix than nicked or linear plasmid.
凝胶萃取
凝胶萃取is performed to recover DNA fragment of interest from an agarose gel. The first step is to excise the agarose gel containing DNA fragment of the desirable size. The gel is then placed inside a purification column that contains silica membrane. Then, binding buffer containing chaotropic agent is added to dissolve the agarose gel, denature protein, and promote DNA binding to the silica membrane. In order to enhance the gel dissolving process, the mixture is incubated at 55-60oC. Other remaining residues are removed using the wash buffer. The last step is the addition of the elution buffer to elute the purified DNA from the purification column.