Today resembled Friday. A re-run of Bertin’s NCP aggregation was attempted, however, results did not resemble Bertin’s. Large concentrations of Magnesium were approached in the NCP solution, however, no significant NCP was observed to be aggregating. One suggested problem was the sample was old and poorly handled, resulting in the essential tails of the NCP being degraded. Next week a better sample will be used.
Today, we repeated the same experiment we did last time in which we added MgCl to the solution of NCP so we can observe the reduction in concentration but we did not observe that. We decided to then make a new solution of NCP from chicken blood starting tomorrow and then we will repeat the experiment so we can spot out where we went wrong with it.
With yesterdays data in disagreement with Bertin’s experiment, today was used to determine the reasons for these discrepancies. We began by adding surplus amounts of Magnesium to the solution in an attempt to see some form of NCP falling out of solution. This was to no avail as the solutions continually tested a similar concentration of NCP from UV vis spectrometer testing. The reasons why this experiment is not consistent remains unknown. Perhaps the Magnesium was incorrectly concentrated. The experiment may need to be performed again.
We added more magnesium to the NCP solution to see if aggregation occurs but after testing the concentration of the NCP after doing that, we found out that the concentration remained the same.. However this is not meant to be so. The addition of the magnesium salt should decrease the concentration of NCP in solution. We will look into this on Monday
Today was spent attempting to simulate Bertin’s NCP aggregation experiments. Magnesium was repeatedly added to a 1mg/mL NCP solution at the increments indicated in the previous blog. Unusually, no significant change was observed in the NCP concentration as a result of Mg2+ addition to the level of 15mM. Futher investigation into the reasons behind this disagreement with Bertin’s experiment will be perused tomorrow. The experiment may have to be repeated.
Today, we went ahead to perform the experiments with the solutions we prepared yesterday. We added amounts of magnesium to the NCP solution and then tested the concentration of the NCP in solution after each addition with the UV based spectrometer. The expected result was for the concentration of the NCP in solution to decrease because the magnesium should make some of the NCP aggregrate and fall out of solution. However this was not the case.So we decided to add higher concentrations of magnesium ton the NCP solution tomorrow and see what happens.
Today I looked over the results from yesterdays test. This time, I found that none of the samples contained magnesium, which is different from what the dilute series showed. Everything about this first test seemed very solid, so I made three more concentrated series to test. Together, these four series should provide a very accurate look as to how the ions behave in these samples. I will be able to compare them more tomorrow.
Today I spent a very large amount of time making a very accurate calibration series. This involved double checking all of my solutions with an accurate scale to make sure the pipettes were being sufficiently accurate. Using this, I was able to able to measure very accurate amounts of the stock solutions, and make a much better series. In the afternoon, I made a concentrated set of DNA samples and ran them with my new series.
Today, we prepared solutions in order to try to replicate Bertins experiment in which increasing concentrations of Mg are added to the nucleosome solution to see how the nucleosome aggregraates at the different concentrations.First we made a 10ml of a 1M solution of Magnesium chloride. Then we prepared the nucleosome solution by adding 67.2μL of the concentrated nucleosome to 10μL of 1M tris and then diluted the solution to 100μL with DI water. It is this solution we will add the magnesium chloride to while testing the concentration of NCP left in the solution after each addition using the UV based spectrometer shown above
Today was spent creating a protocol for the Bertin experiment and setting up the experiment, so come 9am the work can be pushed through. The experiment will consist of a 1mg/mL solution of NCP particles being immersed in an increasing concentration of Magnesium solution. The 12 different concentrations that will be measured and compared to the concentration of NCP particles will be approximately .2mM, .4, .6, .8, 1.3, 1.9, 2.6, 3.7, 4.9, 6.1, and 8.6.
The purpose of the experiment will be to compare the results to Bertin’s results.
