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Today I prepared and ran the first trial of the new series, which was precipitated in 2mM Co and 20mM Mg. Using the approximate concentrations I got from the dilute trial, I also made a new calibration standard. I will be able to analyze the results more tomorrow. I also spent some time today preparing for a paper presentation tomorrow, which I will be making on how osmotic pressure depends on DNA and ion concentrations in solution. They found that at low DNA concentrations, the ions in solution do affect osmotic pressure, but at high DNA concentrations, the concentration of DNA contributes more to the osmotic pressure.

I won’t beat around the bush – Today was a lesson on why undergrads should not compound their mistakes. It began well with all processes inline to finalize the extraction of nucleosomes. However, I allowed the beaded solution to run too long, and it dried out. So at the beginning of the 3 hour processes to restore the solution, the delicate tube was cracked, setting us back even further. The only consolation was that the separation of the DNA experienced a set back, so there will be a chance to catch up tomorrow. Until then, time to read. Foreign factor out.

Today , things did not go as planned. First we discovered that the separation beads we left in the cold room had dried out. We had left the beads in the cold room with the intention of getting it ready for the NCP that was currently running through the gel. So we decided to re-run the beads but in doing that, we broke the tube in which the beads were in. As we quickly tried to retrieve what we could of the beading solution, Professor Andresen told us that the gel did not run properly and we would have to repeat it. This means that we have to wait till tomorrow to proceed with the NCP procedure. Tomorrow will be a better day!

Today, we continued with the extraction of the NCP from the chicken blood. We were able to get about 15mg in total and now the solution is sitting in an apparatus that uses gel to separate the DNA into base pairs with differences of 10.

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.