Today we continued the NCP prep. We found we had recruited around 15mg of NCP particles from the 3ml of chicken blood. Part of the NCP solution was then processed using different techniques to eliminate the protein core and is currently sitting in a gel apparatus that separates DNA chains by lengths of 10bp increments. Tomorrow the process should be completed.
Today, we kicked off preparing a new sample of NCP right from scratch using the red blood cells from the chicken blood gotten from last year as the source. We decided to do this because of the abnormal test results we got from using the NCP solution from last year. A suggested explanation for the weird behavior in which the NCP did not aggregate was that the sample may have been mishandled which led to its damage. So we started with the long process of separating the NCP form the cells. We started of with the erythrocyte lysing, then the nuclei lysing and finally the nuclei digestion. We ran out of time mid way through the process but we will hopefully finish up tomorrow. Pictures of solution at different points of the extraction are shown above.
Today I prepared and ran the other three trials of this second Tris and Mg series. When looking at the averaged results, there are several important things to note. First of all, there are several strange outlying points. In one sample, there was an abnormally large amount of magnesium. In two others, a strangely large amount of DNA precipitated, but there was little change in the ion concentrations. I will be looking more into what caused these random points. In general though, the results are very consistent and show an interesting pattern. As the PEG concentration is increased, the amount of DNA slowly decreases, and then towards the end it rapidly decreases. The Co and Mg concentrations stay fairly consistent over the range, which leads to a huge spike in contributed charges at the higher PEG concentrations. For the most part, the contributed charge by both ions adds up to around one, but when the spike occurs, the total goes up to above two. Some of this strange behavior could be coming from the comparatively large concentration of Tris?
Today I prepared and ran a trial of the next series, which contains magnesium and Tris as well as cobalt. This also involved making another calibration series, with a slightly smaller range than the last one. These different ions in the solution should lead to some interesting effects.
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
