Introduction: In this lab, I worked with Nikhil Patel, Jackie Gibson, and Kellen Mayberry. The purpose of this lab was to get an introduction to the techniques and develop the finesse required to perform DNA labs. In preparation for this lab, Dr. Shingleton taught us how to use micropipettors. While using these instruments, I was simply amazed that it was possible for me, a clumsy teenager, to be so precise in the amount of sample I needed to use. In addition to introducing us to micropipetting, this lab taught about electrophoresis. Electrophoresis is important to DNA analysis labs because it separates macromolecules according to their size and charge. Using electrophoresis, scientists are able to analyze samples of DNA, RNA, and proteins. We used electrophoresis to analyze and identify the dyes that color M&M’s. Materials and Methods: To start off, we picked an M&M and placed it into a cup. (I picked green). We then added .5 mL of dye extraction solution to the cup and swirled it around until almost all of the dye came off of the M&M. After removing the M&M, we micropipetted the dye out of the cup and into microcentrifuge tubes. We then centrifuged our samples in order to separate the solution from the dyes. We then placed our samples into the refrigerator. We then created 4 four standards to run alongside our gel. These standards were prepared according to the table below. We then placed all of our samples into an Eppendorf rack, shown below. One thing that we unfortunately forgot to do was label our own M&M samples clearly. We decided rather the remember what color was in the tube and the position of the tube. This could have led to confusion, and is definitely something I will remember to do in the future. We also created our gel, using melted 1% agarose. We wrapped electrical tape around the gel rig and placed the comb in the middle. After this, we had Dr. Shingleton carefully pour the gel in. Once the gel was set, we tried to move the gel to the electrophoresis chamber and had an incident. We learned the hard way that gel cannot survive gravity induced trauma. The gel unfortunately fell and broke, leaving only 3 of our wells still intact. Because of this, we were not able to fully analyze our data and had to use the data of our classmates. Despite the damage done to our gel, we still loaded it in order to get the experience and develop the finesse required for our later labs. After loading the gel and setting up the rig, we put the lid on and connected the power. We ran our gels at 100 V for 15 minutes. After running our gel, we placed it on a UV light so that we could see the results. Results (Results taken from Jack Grossman, Liam Shingleton, and Olivia Hicks due to the fact that agarose gel cannot survive gravity induced trauma.) Conclusions
Using the data from the other group, I can see that Yellow 5 dye traveled the furthest, while the Blue 1 dye traveled the least. This tells me that the Yellow 5 dye is more negative and is a smaller molecule than Blue 1 dye. I can conclude this because agarose gel is like a sponge, allowing smaller particles to travel through it more easily. I know both dyes are negative because they traveled in the same direction, but Yellow 5 is more negative because it traveled further. Based on the data, it is probable that t Blue M&M contained the Blue 1 food dye. The a Green M&M contained yellow 5, and according to the group I got the data from, it might be possible it contained Blue 1 but it might be due to a spill. The Orange M&M contained either Yellow 6 and/or Red 40. It was hard to tell what it could have contained due to the lack of separation of the dyes. One thing that could have benefitted the experiment is if it were possible to create larger, more separated gels that could run for longer times. Using those larger gels, it would be easier to see the separation between dyes and also limit any cross contamination that could have happened. Discussion The gel we used in our electrophoresis is agarose. It is commonly used in the DNA research world because it is very effective at acting as a “net”, allowing small molecules to pass through it faster. This is due to the fact that when it is more like a solid, it is a porous matrix. This allows for separation between molecules with different molecular weights., measured in Daltons. For example, if there were DNA molecules with weights of 600, 1000, 2000, and 5000 Daltons, the 600 Dalton molecule would travel the farthest because it has the least molecular weight. Gel electrophoresis also separates and moves the molecules using charge. There is a positive and negative end of the gel, and depended on the charge of the molecule, it could migrate either way and any distance depending on its charge. In our lab, we only used 4 common food dyes, however, many others exist! They include the ones shown below. Sadly, we could not have used Betanin, Citrus red 2, and Carminic acid because they have no formal charge, and thus, would not move in our gel. We could use Fast green FCF because it has a negative formal charge and would move in our gel. Gel electrophoresis is a fascinating tool that scientists can use in forensics, genetics, and biochemistry. After doing this lab, I feel prepared to take on what the rest of Genomics class throws at me! Works Cited Freeman, Mary. “What Can Gel Electrophoresis Be Used For?” It Still Works | Giving Old Tech a New Life, itstillworks.com/can-gel-electrophoresis-used-5122149.html. “Gel Electrophoresis.” Science Learning Hub, 20 Nov. 2007, www.sciencelearn.org.nz/ resources/2029-gel-electrophoresis. “Introductory Gel Electrophoresis.” Carolina Biological Supply Company, 2005. Song, Guo Guo-qing, and David Douches. “Agarose Gel Electrophoresis.” 2009.
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AuthorAllan Kalapura. Holland Hall class of 2019. Archives |