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.
Today, we went to check on the nucleosome solutions we left to filter in the cold room and found out that they were in good condition. We then sorted out the collected samples by figuring out which test tubes had the single and double nucleosomes in them. We then tested the samples containing the single nucleosomes to find out the concentration. We did this using a UV based spectrometer which I will show in my next post. After this, we concentrated the sample by using a centrifuge running at 5000g to collect only the nucleosomes from the solution. We tested the concentration of nucleosomes in the concentrated sample and found out that we lost roughly about 7% of the nucleosomes in the solution probably due to the filter in the centrifuge. But over all it was a good result.
Today I spent the entire day looking at the results that I measured yesterday. There was a lot of information there, and it took some time to modify my spreadsheets from last summer. Two of the series displayed clear patterns, and one of them seems somewhat random. The series with a large concentration of Tris has a clear spike at higher concentrations of Tris. The series with a large concentration of Mg has a clear spike of Mg at lower concentrations of Tris. These patterns should be interesting to investigate with future trials. Tomorrow I will be making what will hopefully be a very accurate calibration series to run some concentrated trials.
Today I spent the morning reading several articles on osmotic pressure. I am trying to get a better idea of the results I should expect from the new PEG series I am running. In these tests, the main variable that we will be testing is osmotic pressure. The best idea that I can build as of now is that osmotic pressure has to do with the concentrations of solutes in a solvent. It has several different definitions, depending on the context in which it is being used. The one that seems most relevant to our work is the energy density of solvent molecules. The idea that I am getting is that PEG takes up space in solution that used to be available to water.
I spent the afternoon running my three dilute series after the nitrogen arrived. I will be able to analyze these tomorrow.
Today we revisited last years NCP samples, from Xiangyun Qiu. The day began with a nervous visit to the cold room where we found a good set of UV data from a sample of NCP solution that was left to separate the night before. The picture to the right shows this sample, where particles in a solution were separated through a beaded solution by size. The high spike corresponds to the solution that contained the NCP. Using this graph we were able to pull about 50ml of NCP from the sample, a tone for the experiments we will be running in the future.
From here we centrifuged the NCP solution to create a highly concentrated solution of NCP. Tomorrow we will be using the sample to recreate Aurelie Bertin’s experiments of observing the changing concentration of NCPs in varrying cation (Mg) solutions.
