During gel electrophoresis, applying 130 volts of electricity through the water (or buffer) can cause drastic pH changes. Because of these pH changes, a buffer (a substance that prevents large pH changes) is required. However, several different combinations of the buffers and gels can be used, and each should be tested to determine which is best. This experiment was done to determine which of three buffer and gel combinations is best for using gel electrophoresis with bromphenol blue. Three different combinations of gel and buffer were tested.
They were: (1) H 2 O gel with 1 xTBE buffer, (2) 1 xTBE gel with H 2 O buffer, and (3) 5 xTBE gel with 0. 1 xTBE buffer. The gels were observed over approximately thirty minutes while the voltage was applied. The gels were observed for movement of the bromphenol blue, both from one end of the gel to the other, and from side to side (blurring).
When the negatively charged bromphenol blue was added to the wells in the gel, it sank into the wells to different depths depending on the negative ion concentration in that particular gel. The greater the concentration of ions in the gel, the less the bromphenol blue sank into the well, and vice versa. As the electrical current was applied, both the movement of the bromphenol blue down the length of the gel and it’s dissociation outward was observed. The movement down the gel was caused by the negative bromphenol blue moving toward a small number of positive ions that formed as a result of the current. The dissociation of the bromphenol blue was caused by too much positive charge forming, attracting the bromphenol blue away from the positive ions that were pulling it down the gel. These pockets formed when the buffer cannot supply enough OH- to keep the solution at a constant pH and keep the positive charge pockets from forming.
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In combination (1), the bromphenol blue moved the most down the gel, but also dissociated the most. This meant that the buffer (1 xTBE) worked well to allow positive ions to form to pull the bromphenol blue down the gel. It did not, however, work well to keep the pockets of charge from forming. In combination (2), the bromphenol blue sank the most. This showed that the gel (1 xTBE) had the lowest concentration of ions in the gel. In Combination (3), the bromphenol blue sank into the wells the least, moved down the gel the least, and dissociated the least.
This showed that the gel (5 xTBE) had the most ions in it, preventing the bromphenol blue from sinking. It also showed that while the buffer (0. 1 xTBE) was good for preventing pockets of charge from forming, it also prevented the small amount of positive ions necessary for movement down the gel from forming. Overall, (1) moved the most down the gel, but since it dissociated, (1) is not a good combination. (2) also dissociated, and (3) moved the least, making these combinations bad as well.
In order to make this lab more informative, more combinations of gel and buffer should be provided to allow more a more complete final comparison. Also, a base combination to compare it to should be provided. Some error may also have occurred in delivery of the bromphenol blue because of lack of experience. Some practice should be done before the actual experiment.
Finally, more time should be allowed to understand the experiment to it’s fullest and to ensure that it is done correctly.
