Early in the 19th century, scientists worked with the basic principles of electrophoresis. Scientists measured the behaviour and properties of small ions moving through solutions in an electric field. In the 1930’s, Arne Tiselius developed the “Tiselius apparatus” for separating protein molecules through electrophoresis. By the 1960’s, many different gel electrophoresis methods made it possible to separate different molecules, driving the rise to molecular biology.
Gel electrophoresis is the process by which molecules can be separated by size and electric charge through the application of an electric current. The strength of the current moves molecules through the pores of a thin layer of the gel, a firm jelly-like substance. The gel can be made so that its pores are only the right dimensions for separating molecules with a specific range of sizes and shapes. Smaller fragments usually travel farther than larger ones. In this experiment, a gel was used to separate the six tracking dyes; Bromophenol Blue, Crystal Violet, Orange G, Malachite Green, Xylene Cyanol and Dye Mixture. The gel that was used to separate the tracking dyes was agarose gel.
In the first part of the lab, the agarose gel was poured into the tray while a gel comb was inserted in the center and was rested for twenty minutes until the gel partially solidified. The comb was then removed from the gel and the gel ended up with eight wells. Each well was assigned with a specific tracking dye. After the tracking dye was placed into the wells, the tray was placed into an electrophoresis chamber which contained 1X TBE running buffer. Once the trays were in the chamber and the chamber was covered, the machine was plugged into a power supply and was set to 110V. After approximately 10 minutes, the tracking dyes started to move towards the negative poles and the positive poles of the tray. Therefore, the purpose of this investigation was to separate the tracking dyes and determine their molecular charge. The experiment displayed how fragments were separated based on their size, which in turn affected how fast they were able to move through the gel.
This experiment will help us have a better understanding of the process and the purpose of the gel electrophoresis. In addition, this experiment will provide us more knowledge on how DNA reacts in the process of gel electrophoresis and it will also determine the polarity of the tracking dyes.
Gel electrophoresis is the process in which molecules can be separated by size and polarity by the application of an electric current while the molecules are in the gel. In the 1930’s a scientist named Arne Tiselius changed the world of science with the discovery of electrophoresis (The Columbia Electronic Encyclopedia, 2004). Before this invention was represented, the basis of DNA separation was already discovered. In the 1800’s, Wilhelm, Nernst and Kohlrausch performed an experiment in which the measurement of minuscule ions’ properties was involved as they made their way through a variety of solutions treated by electrical fields (Smithies, 2009). Kohlrausch used the some of the experiments to determine equations for all concentrations of the charged particles that went through the solution.
In 1937, the Tiselius Apparatus was created and developed by Arne Tiselius (The Columbia Electronic Encyclopedia, 2004). The Tiselius Apparatus was an important factor to the rise of electrophoresis, however, it was not able to completely separate the substances that looked like each other until the 1940’s. This was due to the fact that the gels and solids tuned out to be commonly used as mediums instead of the traditional solutions.
Since the 1940’s, major changes have been made to electrophoresis which lead to a variety of new methods. Today, there are numerous gels that are used, for instance, Agarose, Polyacrylamide and Starch. The gel that was used in this experiment was agarose gel because of its easy casting and the simple recovery of remaining fragments. Agarose gel was derived from pure seaweed and it was produced by Joe Sambrook, Bill Sudgen and Phil Sharp, in the 1970’s (Smithies, 2009). The dyes that were used in this experiment were; Bromophenol Blue, Crystal Violet, Orange G, Malachite Green and Xylene Cyanol.
Bromophenol Blue (C19H10Br4O5S) is used as an acid-base indicator, a color marker and a dye. It is prepared by adding excess bromine to a hot solution of phenolsulfonphthalein in glacial acetic acid. Since the dye has a large negative charge, a molecular weight of 670g/mol and migrates at the same rate of a 150 base pair DNA molecule, it is predicted that it will shift slightly to the positive end (The PubChem Project).
Orange G (C16H10N2Na2O7S2) is generally used to stain keratin, it is also a major component for pollen staining. Since it has a negative charge and has the same migration rate as a 50 base pair DNA molecule, it is hypothesized that it will shift far into the positive end (Sigma-Aldrich).
Malachite Green (C23H25ClN2) is traditionally used as a dye for materials such as silk, leather, and paper. It is prepared by the condensation of benzaldehyde and dimethylaniline to give leuco malachite green. Due to its positive charge and low molecular weight (364.911 g/mol), it is predicted that it will shift dramatically to the negative end (Canadian Food Inspection Agency)
Xylene Cyanol (C25H27N2NaO6S2) is commonly used as a gel electrophoresis size marker. It With its smaller molecular weight of 539g/mol, its negative charge and its migration rate equivalent to a 750 base pair DNA molecule, it is predicted that xylene cyanol will proceed far into the positive end (Sigma-Aldrich).
Crystal Violet (C25N3H30Cl) has antibacterial, antifungal, and anthelmintic properties. It was also formerly used as a topical antiseptic (Sigma-Aldrich). The dye can also be prepared by the condensation of formaldehyde and dimethylaniline. Due to its high positive charge, it is thought that crystal violet will travel into the negative end (Sigma-Aldrich).
The purpose of this experiment was to separate six individual dyes from one another and ultimately, to determine the charge and molecular size of the six substances. To do this, the six dyes were subjected to electrophoresis.
A) Casting an Agarose gel
1. The dams were snapped onto each end of the casting tray.
1. The 8-well side of the gel comb was placed into the centre slots of the tray.
1. The agarose was heated up on a hot plate in a 500mL beaker filled with water.
1. 25mL of the melted agarose gel was poured into a graduated cylinder.
1. The agarose in the graduated cylinder was emptied into the gel-casting tray until it attained a depth of approximately of 3 mm.
1. The gel was allowed to solidify for 20 minutes while the gel tray or comb were not disrupted.
1. After 20 minutes, the comb and end dams were carefully removed.
B) Loading and Running
1. 10 µl from each of the listed substances were placed into their respective wells using a micropipette.
Lane 1: Bromophenol Blue
Lane 2: Crystal Violet
Lane 3: Orange G
Lane 4: Malachite Green
Lane 5: Xylene Cyanol
Lane 6: Dye Mixture
1. The loaded gels were placed in the centre of the chamber.
1. Approximately 200mL of 1XTBE running buffer was added to the solution slowly until the level was just above the gel. The buffer was carefully poured into the side of the chamber ensuring that the buffer was not poured directly over the gel.
1. The cover was placed in the electrophoresis chamber.
1. The red patch cord was connected to the red electrode terminal while the black patch cord was connected to the black electrode terminal (refer to figure 1 below for setup).
1. After checking if it was safe to do so, the power supply was plugged in and set to 110V when turned on.
1. The migration of the samples was observed until the fastest moving band reached the end of the gel.
1. After waiting 10 seconds, the patch cords were disconnected from the power supply and the cover was carefully removed.
1. The tray was carefully removed from the gel box and observations were noted.
Table 1: The table below shows the direction of migration of the dyes after conducting gel electrophoresis
Figure 2: When electrophoresis was conducted, the six different dyes migrated different ways
The observation chart (refer to table 1) states the results that occurred in the experiment. The dyes from top to bottom were Bromophenol Blue, Crystal Violet, Orange G, Malachite Green, Xylene Cyanol, and the dye mixture (refer to figure 2). Results show that the malachite green had the least migration on the negative end of the tray while crystal violet had the most. On the positive end, Xylene Cyanol migrated the furthest while Orange G slightly moved. In between Xylene Cyanol and Orange G was Bromophenol Blue. Since the dye mixture is a mixture of all the other dyes, some components of it were observed to travel towards the anode, while others travelled towards the cathode.
From analysing the migration of the different dyes in the wells, many observations could have been made using the data obtained. At the end of the experiment, once the gel was taken out from the electrical current, it was observed that the molecules were no longer in their starting point but instead they had migrated to their respective zones on either end of the tray. When an electrical current passed through the substances, their attractive force pulled them at different speeds depending on their individual characteristics. It was notable that all negatively charged dyes migrated towards the positive end and all the positively charged dyes migrated towards the negative end. Therefore, one common property of the dyes is that they are attracted to the opposite charge. Therefore, the hypothesis was confirmed.
In the science world, electrophoresis is used to separate DNA fragments by size. When the phosphate backbone of a DNA strand is put through an electrical current, the DNA fragments move towards the anode due to its negative charge. The gel electrophoresis creates pores which allows the DNA fragments to migrate to the positive end of the tray. During this process, the smaller DNA fragments make their way through but the large fragments meet more resistance and they end up at different locations.
The observations from figure 2 showed that some dyes were positively charged, while others had a negative charge. The negatively charged dyes were Bromophenol Blue, Orange G, and Xylene Cyanol. These dyes were negative because after electrophoresis, the dyes had left their wells and had travelled towards the cathode. On the other hand, Crystal Violet and Malachite Green had migrated towards the anode, proving that they were positively charged. The dye mixture consisted of both negatively charged dyes and positively charged dyes because the mixture had left the well and migrated towards both the cathode and anode.
Orange G was the dye that travelled the farthest from the wells to an electrode. This migration can be explained by its small molecular size. Since it had a small molecular weight (429.4 g/mol), the molecules were easily able to get farther down the gel, through the agarose gel’s pores. The large molecular weight of Xylene Cyanol (566.71 g/mol) and Bromophenol Blue (670.0 g/mol) explains why the dyes travelled the least from the wells (Sigma-Aldrich). Since the concentration of the agarose in the gel was 2.0%, the pores were very small. Because of this, many dyes had difficulty moving through the gel, which resulted in blurry bands.
The dye mixture consisted of Orange G, Bromophenol Blue, Malachite Green, and Xylene Cyanol. These 4 different dyes were in the dye mixture because of the evidence of the different colours in lane six (refer to figure 2). These four dyes can be seen on both sides of the tray. Orange G travelled the farthest down the well, followed by Bromophenol Blue and Xylene Cyanol. The order of the dyes corresponds to the charges and molecular weights of the dyes. The dye mixture also had Malachite Green on the negative end.
Although the results of the experiment were consistent with those of peers and scientists, a source of error could have been caused when loading the gel. If there was too little of a dye loaded into the well, that dye would be able to easily able to travel through the gel compared to a fully loaded well. The smaller volume of the dye would create a result where a dye is shown to have a smaller molecular size than it actually does. If the intensity of the polarity of one gel is stronger than another’s, the dye will travel farther. These factors would make a dye that has the strongest polarity seem like it has a smaller molecular size, causing it to migrate farther.
Another source of error in this lab was the use of 2% concentration of agarose. The 2% agarose created pores too small for the dyes, the lower the concentration, the easier it is for molecules to travel through. The concentration was too high because the dye samples did not go far, they were clumped up together. A lower concentration of agarose is desired for larger molecules because it would result in a greater separation between bands that are close in size. If there was more separation in the bands, the different dyes in the dye mixture could be easily recognized, and the dye that traveled the farthest and had the smallest molecular size could be better identified. The concentrations of agarose in the gel can range from 0.5% to 2%, but 1% is best for most experiments (Douches, 2009).
Adams, S. (2008) “Agarose Gel Electrophoresis.” Retrieved from http://abe.leeward.hawaii.edu/Protocols/DNA%20Gel%20Preparation%20and%20Electrophoresis.htm
Author Unknown (2002). “Bromphenol Blue – PubChem.” The PubChem Project. Retrieved from. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=8272
Author Unknown (2004). “Crystal violet solution” Sigma-Aldrich. Retrieved from http://www.sigmaaldrich.com/catalog/product/sigma/ht90132?lang=en®ion=CA
Douches, D. (2009). Agarose gel electrophoresis. Retrieved from https://www.msu.edu/course/css/451/Lecture/PT-electrophoresis (2009).pdf
Author Unknown (2006). “Malachite Green” Canadian Food Inspection Agency. Retrieved from http://www.inspection.gc.ca/food/information-for-consumers/fact-sheets/specific-products-and-risks/chemical-hazards/malachite-green/eng/1332268890141/1332268947157
Author Unknown (2004). “Orange G” Sigma-Aldrich. Retrieved from <http://www.sigmaaldrich.com/catalog/product/sigma/o3756?lang=en®ion=CA>.
The Columbia Electronic Encyclopedia. (2004). Arne Tiselius. Retrieved from http://www.reference.com/browse/arnetiselius
Smithies, O. (2009) “How it all began: a personal history of gel electrophoresis”. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22585472>
Author Unknown (2004). “Xylene Cyanol” Sigma-Aldrich. Retrieved from <http://www.sigmaaldrich.com/catalog/product/sigma/x4126?lang=en®ion=CA>.
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