Andresen Lab Blog

Today I analyzed the four concentrated trials I ran yesterday. Overall, there were a few interesting points that I found. Within the range of our error, there was no magnesium in any of the samples, which makes sense because none was added to begin with. There was a fairly consistent amount of Phosphorus (DNA) in all of the samples, which ranged between 500 and 600 ppb. In general, as the concentration of PEG was increased, more Cobalt was precipitated. Logically, this led to an increase in the charge contributed by the cobalt at higher PEG concentrations. At the highest PEG concentration, the charge contributed was around 1, which raises the question what was compensating for the charge at lower PEG concentrations.

Today we departed from the frustration of testing the aggregation abilities of old nucleosome samples and started from scratch. We are currently practicing the technique of NCP prep, or in other words extracting the nucleosomes from chicken blood. The process takes over a day, today being spent using a tris, NaCl, EDTA and PMSF solution and centrifugal techniques to reduce the chromosomes. However, an overnight process if chromatin dialysis is essential. So we will pick un tomorrow with chromatin.

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.