Agarose gel electrophoresis is a laboratory method used to separate DNA or RNA fragments according to size. The principle behind this method is that DNA and RNA, which are negatively charged because of their structure, will move towards a positive charge through pores in the gel when connected to an electric field. Smaller fragments move faster compared to bigger fragments. The presence of the DNA/RNA of interest is then determined by visualising this gel under a gel documentation system. With the DNA/RNA illuminated, usually by UV light, the size of the pieces can be compared to that of a molecular ladder to estimate the length of the fragments. For my first gel, I aimed to detect rotavirus-positive samples from stool samples collected from children with suspected rotavirus infections. PCR amplification was first performed using primers specific to the target DNA. The gel was run to detect positive samples using the separation of the DNA relative to a molecular DNA marker.
I learned a few tips during this experiment which I believe will help fast-track my next analysis that I will perform using this method, and hopefully yours too. Here are my tips for successful gel electrophoresis the first time:
1. DNA/RNA bands will not always be seen on the first run: The protocol for running a gel may not seem complex, which could make anyone using it have high hopes of getting results on their first run, however this is not always the case. Just like optimisations are necessary for every laboratory experiment, running a successful gel requires optimisations too. By optimising each of the reagents using my positive and negative controls, like the agarose gel density, running buffer type and concentrations, and DNA visualisation technique, I was finally able to find the right combination of reagents to obtain the best bands for my analysis.
Lesson: Perform optimisations with your controls before processing your experimental samples.
2. The size of the tank and gel matters: At the start of my experiment, I had the option of choosing between a 1.5L volume tank and a 0.5L volume tank, with a larger and smaller gel required, respectively. I chose the 1.5L tank. I did not think it mattered in the beginning until midway through my experiments. I had to perform at least 10 gels during the optimization before I could finally run my experimental samples. During this time, I used 1.5 litres of running buffer for each gel, and I did not have an unlimited supply. After my fifth run, I noticed my supplies were halfway gone. The visualisation dye was calculated per volume of buffer so the more buffer I had, the more dye I used. Since the optimization was performed using just controls, I had extra wells on the big gel which I did not need. For the subsequent runs, I chose to switch to the 0.5L tank to save ample reagents.
Lesson: The bigger tank and gel were more useful when I had to load more samples, otherwise, the smaller tank and gel helped me maximise my reagents. Choosing the correct size set up is critical.
3. Check your charge orientation: One common, although seemingly silly, mistake to make when running your first gel is to forget to check the orientation of the positive (typically red) and negative charge (typically black) wires on the gel box or tank. If the gel is run from positive to negative, instead of negative to positive, you run the risk of literally running your samples right off the top of the gel! This can happen to the best of scientists. To avoid this, it is best practice to always check that the negative charge wire (black) is plugged into the top of the gel box and the positive charge wire (red) is plugged into the bottom of the gel box. Be sure to check that the wires are plugged into the correct locations on the power supply as well!
Lesson: Make sure to run your samples on the gel towards the positive charge. A.K.A. “run to red”.
4. Running the gel is not the last step in the process: After I ran my gel, I thought all the hard work was done but I was wrong. I needed to visualise this gel to be able to make any meaning of the results. Although the position of the sample loading dye is used to track the progress of the gel, it has no meaning when trying to locate the DNA/RNA bands. These had to be visualised under UV using the gel visualisation machine. Finding out how to use the gel visualisation machine before the gel is complete is very important. For the first run of my gel, I had to discard it because I had not planned for the visualisation. The software was not up to date and the settings were not checked in a long time.
Lessons: Plan for other aspects of the gel electrophoresis, like the visualisation step, not just for running the gel.
5. Reading the gel is not as easy as it seems: Joy comes when two things can be seen on the gel, that is, the molecular ladder and the positive control DNA/RNA sample. This tells you that your gel is ready for analysis. Reading this gel is, however, not as easy as it seems. This can be especially true with the presence of primer dimers. Primer dimers are short DNA fragments amplified when the forward and reverse primers in PCR bind to each other rather than the target DNA to amplify. In cases where you have very small band sizes to analyse, it becomes difficult to distinguish between primer dimers and the positive samples. This problem can be solved by using the appropriate molecular ladder or extra PCR optimization.
Lesson: Choose a molecular ladder according to the size of your target bands. Do not use a 1kb ladder for fragments that are 100 bp in size.
Overall, gel electrophoresis gave me a better understanding of my work compared to the analysis I did with other methods. I spent four months optimising my reagents because I had not expected some of the challenges I faced. I underestimated the importance of optimisation and did not consider that in planning my work. In the end, I learned a lot during the analysis, and I hope these tips help you in running your gel experiments.
A little bit about guest writer Flavia Kaduni Bawa:
Flavia is a PhD Candidate at the West Africa Centre for Cell Biology of Infectious Pathogens at the University of Ghana. Her current work focuses on evaluating the potential of a host-
response assay to distinguish between malaria, viruses, and bacteria as causes of childhood
febrile illness. She is passionate about communicating science to aspiring scientists and people without a science background.