Today we looked at the latest results of the NCP experiment. These came out much better, and the concentrations were extremely consistent. They showed some interesting trends, mainly that there was a range where the Mg compensated for all the charge of the nucleosomes, but did not cause them to precipitate. Our main problem now is the large amount of uncertainty we are seeing. We will try to drive this uncertainty down with future tests.
Today we analyzed the data from the NCP experiment. The results are still very inconsistent, and are not showing any clear trends. They are showing some inconsistencies in all of the concentration values. Because of this, we prepared for another test this evening, which we can look at tomorrow.
The results we got from running the two buffers where a little inconclusive due to some issues with the magnesium concentrations in the buffer I made and some other complications with Ben’s buffer.But,we could still see the precipitation pattern of the NCP as the MG concentration increased. So Professor Andresen decided we needed to repeat the experiment to find out what as going wrong with it. However, this time, we spun the NCP eight times with the buffer because Professor Andresen suspected that the four spins we did for the last three experiments were not enough. We will analyze the results tomorrow and hopefully we get conclusive results this time.
Today we had a pretty big day. We ran two sets of NCP experiments, which involved running the machine well into the night. We are having some problems with inconsistent Mg concentrations, which we hope to solve with future trials. We will be analyzing the data tomorrow, as well as preparing future trials.
Today I ran two trials of the buffer solutions for the “5mM” Cohex series. The results leave me a little skeptic that the DNA was actually precipitated in 5mM, because all of the buffer solutions contained pretty exactly 3mM. This would explain why the Mg values only range from 0 to 50, which was the same range used for the 2mM series last summer. I will look into the other buffer solution later this week, to see if it contains 5mM or 10mM.
Today, we did got some good news and started working towards better news. Professor Andresen got the results of the gel he ran yesterday and found out that we had a good amount of NCP in our sample. Next, we found out that the test we ran with the spectrometer to monitor the ions present in our solutions was somewhat positive. There were a little off-concentrations in the solutions I made but ultimately, we noticed that the amount of phosphorus in the solution dropped steadily as the concentration of Mg in the solution increased. This is good news because it means that the NCP’s are falling out of solution(condensing) as they contain phosphorus. To get better results, we decided to run the same tests again but this time with two different buffer solutions. The same one I made yesterday and a new one containing Tris and Nacl without Mg. We will analyze the results tomorrow.
Today we got the NCPs ready to run in the spectrometer. Last week friday, we had a series of buffers with different concentrations of Mg in it for the eight different samples of NCP we were going to work on.First we spun down the NCP we had in a centrifuge three times in a buffer that contained MgCl. We then collected the NCP from the filter and tested the pH of the NCP to see if the buffer was effective and indeed it was because the concentration remained at 7.0. We diluted our eight magnesium samples, NCP, and and the buffer we used in the third spin by a dilution factor of 60. Then put them in the spectrometer so we could test the amount of Mg ions in each. We will analyze the results tomorrow. Here is a picture of the test paper we used. The yellowish colour represents neutral and as you can see , the first 8 dots represent our 8 samples and the bigger yellow dot represents de-ionized water which is neutral. Since they have the same colour,we can conclude that the buffers where neutral.
Today I took a somewhat short day, with a planetarium show in the afternoon. In the morning, I tried to catch up on some reading. I read one article on the competition of monovalent anions, which was helpful because it tells us we can safely substitute out Chlorine for something that is more measurable. I also got ready to start running the buffer solutions, which will give us an exact look at the solutions in which our DNA was precipitated.
Today I ran the remaining three trials of the 5mM series. These results confirmed that there is something wrong with the second and fourth samples, but other than that they were very consistent. The results were also strangely linear, which we saw some of in our results last summer.
Today, finally finished separating the di-nucleosomes from the mono-nucleosomes as I explained yesterday . Looking at the picture on the right,you can see some peaks on the graph . We are not sure yet which peaks represent what nucleosomes at this point but we suspect that the first pick to your right is where the mono- nucleosomes fall because a test using the UV based spectrometer showed a high concentration of NCP. We will run the different samples at each peak through the gel on Monday and then compare them again to the DNA ladder , so we can finally say for sure what we have.
