The main purpose of the original experiment was not accomplished. During this experiment due to the lack of DNA needed to prepare the 19 solutions with different concentrations over six orders of magnitude and also the BPES buffer, the different absorptions could not be measured and no calculations were possible. As a consequence, the binding constant of Ethidium Bromide to DNA using absorption spectroscopy was impossible to measure since no data was available. Instead, the whole experiment was redesigned by the TA in order to work which what was available at the lab and at least get a sense on how absorption spectroscopy works.
Experiment was performed in partners order to accommodate for the last minute changes in the experimental procedures …show more content…
Solution 1 was made of 10 microliters of stock solution of ethidium bromide plus 1 mL of DI water. Solution 2 was made of 10 microliters ethidium bromide, 1 mL of DI water, and 1 microliter of the purified plasmid pBR322 corresponding to student 1, were delivered with the corresponding micropipette to the tube. The third solution was prepared in the same way as solution 1 but with the only difference on the plasmid used, in this case the plasmid came from the purification experiment performed by student 2. The solutions on the cuvettes 2 and 3 turned from a clear into a light pink color. It is important to mention that these two plasmids used to come from the purification experiment performed a few weeks ago by each of the involved students. Each solution was introduced on the absorption spectroscopy apparatus in order to measure its absorbance and values of 0.307, 0.315, and 0.317 respectively were obtained at a wavelength max of 480 nm as asked by the lab manual used for the experiment. When looking at the three values obtained it can be observed than the values of solutions 2 and 3 where the plasmids were used have values of absorption pretty closed to each other, which makes sense since the purification process of the plasmids results on DNA size
Experiment was performed in partners order to accommodate for the last minute changes in the experimental procedures …show more content…
Solution 1 was made of 10 microliters of stock solution of ethidium bromide plus 1 mL of DI water. Solution 2 was made of 10 microliters ethidium bromide, 1 mL of DI water, and 1 microliter of the purified plasmid pBR322 corresponding to student 1, were delivered with the corresponding micropipette to the tube. The third solution was prepared in the same way as solution 1 but with the only difference on the plasmid used, in this case the plasmid came from the purification experiment performed by student 2. The solutions on the cuvettes 2 and 3 turned from a clear into a light pink color. It is important to mention that these two plasmids used to come from the purification experiment performed a few weeks ago by each of the involved students. Each solution was introduced on the absorption spectroscopy apparatus in order to measure its absorbance and values of 0.307, 0.315, and 0.317 respectively were obtained at a wavelength max of 480 nm as asked by the lab manual used for the experiment. When looking at the three values obtained it can be observed than the values of solutions 2 and 3 where the plasmids were used have values of absorption pretty closed to each other, which makes sense since the purification process of the plasmids results on DNA size