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.
Today, we took a step ahead: getting the NCP to run through the gel almost perfectly. Looking at the picture beside, the first column is the DNA ladder and the bold band at the middle stands for 100 base pairs. But we need about 150 base pairs so we can count the bands after the 100bp band to see the 150bp mark because the bands in increments of 10. The next column stand for the NCP digest for 0 minutes, the next for 5minutes, then 10,20 up till 50 minutes. If you notice closely, there is a thick band on the same level of the 150bp on the 5minute digest column. This is an indication that 5 minutes was the adequate time to digest the NCP.We do not use the other columns because there is a lot of smearing of the whitish band and this indicates over digesting. Therefore we digested our NCP for only 5 minutes, and with the packing ready, we are currently running our NCP through it to separate the mono-nucleosomes from the di-nucleosomes using the size-exclusion chromatograhy technique. This way, the mono-nucleosomes we actually need run faster than the di-nucleosomes through the packing and we can collect them. The NCP has not finished running through but I will keep an eye on it till it does.
Today I looked at the same 5mM Co series, but at a much higher concentration. Using the results I got from the dilute run, I designed a series that should encompass all the concentrations I will be seeing. After running at a higher concentration, the results look much more consistent. Actually, with the exception of the fourth sample, they look very clean and there is clear trend. It resembles the trends we were seeing last summer, but with the Mg taking longer to overcompensate for the Co. Tomorrow I will run three more trials to look at this series closely and get some averages.
Today I spent the day looking at the 5mM Co series, which goes from 0 to 50mM Mg. I was somewhat unsure as to the concentrations of ions that I would find, so I made a very broad calibration series, and a 100X dilution of the samples to test this out. The results were pretty similar to the series I have run before, but with much more DNA. This makes sense, because our collaborator changed his procedure slightly and prepared the samples with more DNA. It is strange that we did not see an increase in the other ions as well, but it may have something to do with the initial conditions we are looking at for this series. Overall, this dilute run was somewhat unreliable, with particularly strange results for the fourth sample. Tomorrow I will use what I have learned about the concentrations to make a concentrated run.
Today, I was handed two new articles to study. These articles had to do with the extraction of the red blood cells and then the NCP form the chicken blood.
The first one was named ” Isolation of histones and nucleosome cores from mammalian cells”. It summarized the extraction of the NCP from mammals but it was related to what we are doing with the chicken blood so I learned a few extra things.
The other article named ” Salt induced transitions of chromatin core particles studied by tyrosine fluorescence anistropy” was actually where professor Andresen extracted our protocol for extracting the NCP from the chicken blood from. So far,I have learned the roles of the different buffer solutions and also the principles behind all the procedure. I will continue with this article and hope I finish reading it tomorrow .
