Blog Feed

In the first few weeks of the class, our instructor, Dr. Ren, gave the class different science topics to research. These topics focused on lab procedures that we would be completing throughout the semester. Myself, along with @rachelatherton12, were assigned to research gel electrophoresis, its mechanisms, and its various applications.

What is Gel Electrophoresis?

Gel electrophoresis is a technique that allows DNA, RNA, and proteins to be separated by their molecular size and charge. The gel mold itself is made from a mixture of agarose powder and a buffer. The amount of agarose that is used in one gel can be changed to accommodate different DNA base pair lengths. A higher amount of agarose powder in the gel mixture will allow for smaller movements. This is useful when analyzing DNA that have very small base pair lengths. A smaller amount of agarose powder in the gel mixture, conversely, will allow for larger movements, which is useful when analyzing DNA that is larger in size.

Why is Gel Electrophoresis Important?

Gel electrophoresis allows scientists to analyze the results of PCR to determine if the cloning was successful. Furthermore, it allows scientists to see visual proof that a setup to a larger experiment (such as developing sample DNA to insert into E. coli bacterium to test for antibiotic resistance in another species) had either succeeded or failed.

What Determines Movement?

There are a few different factors that determine the movement of DNA, RNA, or proteins through the gel. The first factor that determines the movement in gel electrophoresis is the charge of the molecules. The backbone of DNA and RNA is negatively charged. Therefore, these molecules will tend to move toward a positive charge. This is the primary mechanism for how gel electrophoresis is able to occur. The second factor that determines the movement in gel electrophoresis is the size of the molecule itself. Larger molecules will move slower through the agarose gel when compared to smaller molecules. This is the primary way that the gel is analyzed after the electrophoresis experiment has been completed. The final factor that I know of that determines the movement in gel electrophoresis is, as previously stated, the amount of agarose powder in the gel itself. This determines which molecule sizes should be run, with a higher percentage of agarose powder in the gel being better for smaller molecules, and vice versa.

Steps of Gel Electrophoresis

Before discussing the steps to perform a gel electrophoresis procedure, it is worth mentioning that there are different types of machines that can be used to analyze it the same way. Some procedures have a couple extra steps, but most of the set up is universal with some slight tweaks. I will be discussing the procedure to running a gel electrophoresis reaction for the MiniOne electrophoresis system.

1. Prepare the gel. This is done by combining a mixture of agarose powder and TBE Buffer, with the measurements of each depending on the sample size. This mixture is then heated to a boil in a microwave until all of the agarose powder has been dissolved into the buffer. After all of the agarose powder has been dissolved, a DNA stain is added to the mixture. This mixture is then poured into casting trays to solidify over the course of 20 to 30 minutes. These casting trays contain an attachable comb-like piece of plastic that indents wells into the gel. This is how the gel will be loaded with DNA samples later on.
2. Load the Gel. After the gel has solidified, take one of the gel samples and place it into the electrophoresis chamber. The next thing to do is to fill the chamber with a buffer fluid. This is to prevent the DNA, RNA, and/or protein samples from denaturing. After this, use a 12 microliter micropipette to load the samples into each of the wells.
3. Run the Electrophoresis Reaction. After covering the electrophoresis chamber, the reaction is ready to be run. I advise turning on the light that is built into the chamber every five minutes to observe the movement of the DNA samples. Once no more movement occurs, stop the reaction, and analyze the gel. The analysis of the bands is already built into this machine. Leave the cover on, turn on the light, and bands should appear.

Uses of Gel Electrophoresis

Gel electrophoresis separates DNA, RNA, and proteins. This can be used to:

• Visualize bands of a molecular marker to genotype individual plants
• Verify amplification of Polymerase Chain Reaction (PCR) or sequencing reactions
• Check the quality and quantity of genomic DNA after extraction
• Separate DNA fragments to clone a specific band
• Test plants to determine if they had been genetically modified

References

Carroll, John. “Gel Electrophoresis.” Nature News, Nature Publishing Group, 2014, www.nature.com/scitable/definition/gel-electrophoresis-286/

“Overview of Electrophoresis: Thermo Fisher Scientific – US.” Overview of Electrophoresis | Thermo Fisher Scientific – US, 2016, http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-electrophoresis.html.

Robbins, M. 2018. “Gel Electrophoresis Principles and Applications.” eXtension, https://articles.extension.org/pages/32366/gel-electrophoresis-principles-and-applications