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Today, we ran solutions similar to the ones we did yesterday. The only was change was that we diluted the 1,2,4,6,8,10(ppm) solutions to 1,20,40,60,80,100(parts per billion(ppb)) respectively and then calibrated the spectrometer with them. Also , the samples we used this time were some of the DNA samples John used last year.

We also learned more about analyzing the results from the spectrometer. We noticed one point( the 80ppb) on the graphs were out of place. We probably added a little too much of Cobalt in the original 8ppm solutions we made yesterday. Otherwise, every other thing seemed pretty okay.

Today the veteran put the foreign powers to the test by pulling out an old batch of DNA and left us alone to test the ions present in the solution. The foregin fact passed fairly sucessfully with most of the data corresponding to Johns previous tests. One of the calibrating solutions (80ppb) had a out lying concentration of Cobalt, which threw the calibration and results off a little. But all and all a good day, and feeling more comfortable using the equipment.

Today,John “the veteran” made solutions of magnesium, cobalt and phosphorus of unknown concentrations and our task was to use results from the spectrometer to make good estimates of their concentrations. We started off by making calibrations of the same solution varying from 1ppm(parts per million) to 9ppm.

We then ran these calibrations in the machine along sides johns samples so we could use our calibrations to deduct the concentrations of his solutions. This was another opportunity for us to improve our pipetting skills. Ben taught me how to pipette properly after he noticed that I was unknowingly drawing out to much solution each time.

We ended up getting the right answers to the concentrations of johns mysterious solutions.We are looking forward to the next task.

Today we began with a lesson on chemical safety. After that, I gave a crash course of the basic chemistry that is required for our lab work. We then each prepared a set of samples, spanning from 1 to 10 ppm of Mg, Co and P. Using my set as the calibration, we tested the other sets for accuracy, and they were both very close, especially for first tries.

Today I spent the morning brushing up on some reading, looking at a review article done by Bai, et al. This article described research very similar to my own, because they are also using spectroscopy to look at the ion atmosphere around Nucleic Acids. They did not use precipitated DNA though, rather they use a buffer equilibrium technique to preserve the atmosphere around the DNA. They found that the total ions around the DNA almost always was within error of exactly compensating for the DNAs negative charge. They also studied the competition between different molecules with similar charges, finding that bulkier polyions were often less competitive than the alkali and alkaline earth ions. I then spent the afternoon brushing up on my OES skills, preparing and running a test series and calibrations set.

As promised, learning the experimental ropes began today. It began with a crash course in stoichiometry, led by John, the student veteran of the Andresen lab. Once we had recalled the number crunching of high school chemistry, we were sent to the pipettes to create solutions of varying concentrations of Mg, P, and Co. We created these samples so we could be trained on the main machine of the lab, the Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). To quickly summaries what this machine does, it pumps solutions through a high temperature plasma, which blasts the molecules in solution apart and detects the resulting ions by analyzing the discrete wave lengths detected at different regions throughout the machine.
Using the ICP, we can send DNA on NCP molecules that have been bathed in different ionic solutions, we can detect the ions that attach themselves to molecules. It’s important to note that the main error in the experiments comes from pipetting, so the Foreign Factor will be getting lots of practice on that, competitions galore.

Kurt was not around today so John was in charge. We had a session on lab safety early on, to start the day. Jeremy Kumar gave us essential information on how to handle situations gone wrong in the lab and he gave us helpful information about appropriate lab behavior.

Next, John showed us how to prepare solutions of Cobalt, Magnesium and Phosphorus. We learned how to use the pipettes and brushed up our old school chemistry skills in stoichiometry as we calculated concentrations of solutions.

The most exciting part of the day was learning how to actually run the Spectrometer as John lead the way!!

We rounded up reading the articles we had to cover today. We read the “Physics of Chromatin” article by Helmut Schiessel. This article was like an all round summary of all the other articles we read during the week. First , it talked about the structure of the Nucleosomes, then proceeded to say that DNA is packed in a way that the information would be easy to access even though it is packed in a complex way.

Next, we read about how an increase in ionic strength makes the DNA thicker and hydrogen bonding being responsible for the linking between the DNA- backbones and the histones surrounded by the DNA.

The paper also confirmed once more that counter ion release was responsible for overcharging. The paper ended by using a sphere ad chain model to explain the DNA and histone interaction in three different cases.
1) weakly charged
2) strongly charged and
3) physiological condition

With today being the last day of reading and next week the beginning of learning experimentation procedures, it seems appropriate to discus a few of the experiments we have read about during these past few reading days.

With certain aspects of these nucleosome structures fairly ambiguous, such as how they are packed along the genome, investigation into these uncertain aspects of nucleosome function is currently being looked into. The main work we have looked at is the paper, Structure and Phase Diagram of Nucleosome Core Particles Aggregated by Multivalent Cations, by Aurélie Bertin, Stéphanie Mangenot, Madalena Renouard, Dominique Durand, and Françoise Livolant, where they examined the behavior of NCP in various polyvalent ion solutions. The general trend was that when a solution of NCP had polyvalent ions added, the NCP rapidly fell out of solution. However, as more solution was added the NCPs went back into solution until it reached its original concentration, an unusual trend that that could suggest certain processes and structures of chromatin and NCP.

Ben has basically said all we did today at the lab and has given a little lecture on the Necleosome core particles(NCP),but i will add a little to that. I will explain a little about the experiment that was done on the NCP he talked about. As he said already, the NCP has a net negative charge and so the experiment involved adding two different cations ,namely a magnesium ion and a spermidine ion to a solution containing the NCP.These ions were added separately in two different experiments. The behavior of the NCP was observed as they precipitated when the ions were added but also came out in re-dissolution when the number of ions added reached a certain threshold. They used different concentrations of the NCP to see the different behaviors it will have. We learned that, the lower the concentration of the NCP was, the greater the interaction it had with the ions. We will keep you posted, but for now the second half of the international factor is out till tomorrow.