![]() Introduction: The purpose of this lab to teach us the techniques that are necessary for gel electrophoresis. We learned about these techniques by using polar dyes that are found in M&M’s instead of using DNA. In this lab, we also were taught the techniques of using micropipettes and the skills necessary to analyze a gel. Micropipettes are used to measure and dispense small volume of reagents in many parts of chemistry and biology laboratories. Gel electrophoresis can be used to separate DNA fragments. Electrophoresis uses an electric current to separate different-sized molecules, smaller molecules can move more easily through these gel pores. An example of using gel electrophoresis in the science community is that gel electrophoresis is commonly used in plant breeding and genomics for genotyping with molecular markers. The knowledge that we gained from this lab show us what charges these molecules have and what their sizes are compared to the other dyes that were used in our experiment and it teach us what certain colors make up dyes in M&M’s. Learning these skills will helps us understand DNA and how we study it. Methods: We started by making our agarose molds. To prepare the mold for the agarose gel we wrapped the mold with electric tape and then our teacher, Dr. Shingleton, poured the agarose liquid into our molds. We had to let it dry, so while we waited, we extracted the dye off of the M&Ms. I extracted the color brown. We extracted the colors by adding 0.5 mL of dye extraction solution to the cup that had the M&M. We then used a micropipette to take the dye from the cup and to place it in a microcentrifuge tube that was labeled with our initials and the color of the M&M. We also prepared four standards: Tube A which contained Blue 1, Yellow 5, Yellow 6 and Red 40, Tube B which contained Blue 1, Yellow 5 and Yellow 6, Tube C which contained Blue 1, Yellow 5 and Red 40 and finally Tube D which contained Blue 1, Yellow 6 and Red 40. During the next class time our agarose molds had firmed up and ready to be used, so we removed the tape. However, during this time a member in our group accidentally dropped the gel and it broke. Fortunately, three of our wells were in good shape. So after freaking out about the gel for a bit, we put the gel tray in the rig and filled the rig with a buffer solution. Then we loaded the gel by using the micropipette and we ran those three good wells. We used the green dye in our first lane, brown dye in the second lane, and yellow 5 dye in our third lane. After loading the gels, we ran them at 100v for 15 minutes (10:30 to 10:45). After the gel was finished running, we disconnected the rig from the power source and the we removed the tray so that we can put the gel on a UV light to see the gels and to take a picture of it. In the end our gel looked a bit off because of the error before, the dyes on the right ran correctly, but the dyes on the left were merely for practice. Because of the incident of the dropping of the gel, we had to borrow another group’s data for better results (I borrowed data from Vaishnavi, Elaine, and Gray). In the first lane they used Tube A, second lane was Tube B, third lane was Tube C, fourth lane was Tube D, then Vaishnavi’s tube was the fifth lane (green dye), Gray’s tube was the sixth lane (yellow dye), and Elaine’s tube was the seventh lane (orange dye). After micropipetting the dyes from each tube into a gel, they closed the lid of the gel rig and plugged it into the power source for electrophoresis. They ran the gel at 100v for 17 minutes. Results: The data that Vaishnavi, Elaine, and Gray’s group got is represented in the data table. This table shows the wells’ number of dye bands, distance traveled by each dye, and the direction of the migration of each dye based on the electrophoresis results. ![]() Conclusion: In this lab we were introduced to the methods and equipment that are necessary to perform a genomics lab and it taught us skills that we are going to need in the future for this class. We also observed the migration of dye through the process of electrophoresis and discovered that electrophoresis can be used to separate molecules based on their shape, charge, and size. We found the size and charge of the colored dyes of M&M’s and then we compared those dye colors to the standards (Tube A, Tube B, Tube C, and Tube D). Looking at the results of the borrowed data (since out data was no good), I observed that Gray’s Tube (Yellow 5) traveled the furthest at 3.40 cm meaning that it is the smallest molecule. However, the Blue 1 in Vaishnavi’s green dye had the shortest travel at 1.60 cm meaning that it had the largest molecule. Since the agarose gel was very porous, it allowed for the smaller molecules to travel the quickest and furthest and for the larger molecules to travel the shortest and more slowly. Since the molecules traveled towards the positive end of the gel, they are negatively charged. In their M&M samples they had the dyes blue, yellow, and orange. The yellow dye traveled the furthest meaning it was the smallest molecule and the blue dye traveled the least meaning it was the larger molecule. In most of the lanes in their gel, they had one or two bands of dye. However, in the lane in which Tube C was loaded, lane three, there were the most variety of dye bands, there were three bands: blue, red and yellow. These conclusions make me wonder: why do certain colors have bigger molecules, what does this information mean about the dyes itself? Why does having the shortest distance traveled (larger molecule)/having the furthest distance traveled (smaller molecule) mean? In the conclusion, I wish that my group didn’t make the mistakes that we made so that we got to experience the lab first hand, but accidents happen and it was still a great learning experiences. We will learn from our mistakes! ![]() Discussion: The charge affects the migration of the molecule because electrophoresis involves an electrical field which causes the agarose gel to a positive charge on one end and a negative charge on the other end. The DNA molecules will be pulled towards the positive end of the gel since DNA molecules are negatively charged. Even though we didn’t use actual DNA in this lab, the dyes were negatively charged for this lab.The size of the molecule can affect its migration through an agarose gel because if the molecule is big then it is going to travel slow and a short distance, but if the molecule is small then it will travel far and fast. Since the gel is very porous, the smaller molecules will travel further and faster because they will have an easier time getting through the gel. The pores of the gel allow for an exact separation of the molecules. An example of this is if we used this method to separate molecules based on their molecular weight, if we were to separate molecules with 600 daltons, 1000 daltons, 2000 daltons, and 5000 daltons. Dalton is a unit of measurement that measures DNA (1 dalton is equal to 1 AMU). The DNA molecule with the molecular weight of 600 daltons would travel the fastest and the furthest, then the 1000 dalton molecule, then the 2000 dalton molecule and finally the 5000 dalton molecule will come in the shortest and the slowest. With the addition of the four new dyes (Betanin, Carminic acid, Citrus red 2, and Fast green FCF) the Betanin wouldn’t work because it has a positive charge on the nitrogen with cancels out the negative charge of the oxygen. The Carminic acid and Citrus red 2 wouldn’t work since they have no formal charge and they wouldn’t be attracted to the positive or negative side of the gel during electrophoresis. Therefore, Fast green FCF would be the best to use in the lab since it has the most negative charge causing it to travel towards the positive end of the agarose gel. Work Cited:
Robbins, Matthew. “Gel Electrophoresis Principles and Applications.” EXtension, 1 Feb. 2012, articles.extension.org/pages/32366/gel-electrophoresis-principles-and-applications. "Gel Electrophoresis." Nature News, Nature Publishing Group, www.nature.com/scitable/definition/gel-electrophoresis-286. "Micropipette." A Dictionary of Biology. . Encyclopedia.com 14 Feb. 2018 http://www.encyclopedia.com. “Unified Atomic Mass Unit.” Wikipedia, Wikimedia Foundation, 17 Feb. 2018, en.wikipedia.org/wiki/Unified_atomic_unit. “Using a Micropipette." Oeta PBS Learning, PBS Learning, oeta.pbslearningmedia.org/resource/biot11.sci.life.gen.usingmicro/using-a-micropipette/#.WoT_eK2ZP-Y.
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