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On Tuesday, I continued to run the second series of samples, and I finished all but one of the eight trials. I also began to look at the spectrometer’s ability to analyze chlorine in samples. This will be useful because it is possible that the cobalt hexamine ions are not completely dissociating. What this means, is that some of the cobalt ions we would expect to be +3 will actually be behaving as +2. Knowing the exact concentration of Cl would allow us to see if all of the ions were dissociating. Unfortunately, my first results came out very strange, with the chlorine concentrations going negative for a lot of the values.

Yesterday I spent the morning looking through the spectrometers manuals trying to figure out why the results we are getting are so weird. While doing and alignment i put our sample container into its slot and noticed that only a small amount of light was going through the sample compartment and the rest was just going over it. So the professor re-ajusted the holder and I ran another trial on matt and andrews red and purple solutions and got better results than the first time. I also re-ran the collaborators chromatin and got its absorbance to be 3 times what it was when i ran it the first time.

Yesterday, I continued the testing on the second series of samples. I prepared three more trials, and tested four of them. I also began analyzing the first few trials that I have run. I’ve found that the ends of the series, near 45mM Mg have final concentrations that are very low, near 1-2 ppb, which is very hard to measure. While this results can still be used, it is possible that they are inaccurate. I also started correcting and adding more graphs to my report. I am adding some total charge and final concentration graphs. Due to the low concentrations, I am going to look at designing a concentrated trial today.

Today I prepared the next three trials for the second series, and ran two of them. Everything is going well so far. One thing that I am considering is running a trial at a higher concentration. I think it would be interesting to see if only diluting the solution to 50X or 20X had any effect on the results. I think part of the reason that the results are going so wacky for the higher concentrations of initial magnesium is that the precipitated DNA concentration is so low. I think that running it at a higher concentration would help get accurate data for the higher points. Unfortunately, we don’t have enough sample right now to do this several times, so I will have to plan it carefully to conserve sample. We also had our meeting updating everyone on progress, and also describing some of the work we still need to do.

Yesterday, I ran the first trial of the next series. This series goes from 0 mM to 45 mM initial Mg concentration. As I looked at the results from this first trial, I was glad to see it closely resembled the appropriate part of the first set of trials, which went up to 22.5 mM. The last few points, towards the higher concentrations, and further trials should help see what is going on there. I also reviewed my results report for the first series, and updated all of the graphs. Unfortunately, we found an error in how I was calculating the errors in the measurements. Due to the way I uses excel spreadsheets repeatedly, this error was in all of the data I have analyzed so far. I was able to track down all of the repeated mistakes, and I believe my analysis is no more accurate.

Basically in a nut shell we did another trial digestion using .1 microliters of nuclease instead of 1 microliter and we got the best results to date. We took our data and determined that we would get the best samples with a 20 minute digestion so we did a digestion on the rest of our chromatin. We set up our size separator in the cold room and as we were pouring in the chromatin the professor noticed that our tube had dried out making it useless, so we quickly poured our DNA out and lost a fair bit and then took the tube back to the lab to clean it out. However, during the cleaning we didn’t use a Styrofoam sleeve to protect the glass and accidentally over tightened and broke our tube, so now we are at a stand still waiting for new materials. With my free time i have worked a lot on the website and did quite a fair bit of reading. Today I took the scatchard plot of the nucleosomes from a similar experiment to ours and used a program to scale and choose each of the points to figure out what their actual locations are and then turned it into a read-able graph that we will look at on Monday.

A lot has happened since I last posted. Let’s start with the positive.

We have successfully run a very nice non-denaturing poly-acrylamide gel. The key was to increase the amount of sucrose in or loading solutions. This helped minimize diffusion of the samples before they started running. Another thing that helped was a mistake I made. In my haste to make the gel, I accidentally inserted a 10-well comb rather than a 15-well comb. However, this actually resulted in much nicer results.

We see exactly what we would like. The first column is our normal DNA ladder. The second is the undigested chromatin. As you can see, it is all at the top of the gel. Then we start digesting. As we go to the right, the time of digestion is increasing. As you can see, by the sixth column or so, the size doesn’t change much for a few columns. This is the digestion being stopped right at the nucleosome core. After a bit of time, however, the digestion proceeds past this point in some of the nucleosomes, digesting the DNA that is around the core. That is why the gels get smeared towards the bottom for the last three columns. When we do our final digestion, we will make sure that we don’t let it digest this long.

After this great success, we digested one of our samples and were going to pass it through our size-exclusion chromatography column. This column will separate our sample by size. This will allow us to collect all of the nucleosomes that are the exactly correct size, while ignoring those too big or anything that is too small (bits of DNA, etc.). The setup is shown to the right.

One thing about a size-exclusion column is that it must never dry out. The tricky thing is that you need it to almost dry out to add your sample so that your sample stays relatively concentrated in the gel. To make a long story short, I brought the column to the almost dry level and the closed the valve. However, the valve leaked slightly and after 15 minutes, when we loaded the sample, I noticed the gel had completely dried out.

At this point we needed to recover the sample (which we did right away). We then took the column back to the lab to recover the media (the white stuff in the column) so we could rehydrate and repack the column. During this process, I overtightened the column in the vise that was holding it. So now we are waiting on a replacement column and replacement media. It’s funny how much a small leaky valve and 15 minutes can change things.

Today, I had the Spectrometer running the entire day, and I made it through all 4 trials that I wanted to run. So far, I have found all of the results to be fairly consistent with my first set of four trials, which is good news. I think that the new calibration standard may have helped to lessen the extreme errors I was getting with the eighth and tenth points. All together, I now have 8 trials that I can include in my report of my results so far. This should give us a very good idea of the patterns of the ion concentrations. Tomorrow, I am going to start preparing the second series, which is also a magnesium series. The second series goes from 0 to 45 mM Mg. I will also being finishing up my report of my work so far, which I will probably be putting up on here. Travis and I have been putting together a website that summarizes the work that we are doing, which can be seen here: www.gburgsummerphysics.webs.com. This will be a good overall explanation of the theories and processes involved.

Yesterday, I analyzed the final of four initial sample tests that I have been running. It came out to be similar to the other three trails, which is good for consistency. I will now be moving on to the next set of trials for this first set of samples, which I expect to be even more accurate. Using a better calibration standard should make this set of data more reliable. I also tested my new calibration standards, and found that they worked quite well. After diluting each one to the proper concentration, I am left with about 80 test tubes just waiting to be tested, which I should hopefully be able to finish tomorrow.

Yesterday morning, I read two different articles. One was about DNA stretching, and the other was about incomplete ion dissociation. The DNA stretching article was not as relevant as I had hoped, but it was still interesting. It was about a statistical mechanical model for the condensing of DNA under tension. It was based on data similar to that which I am collecting, on the condensing of DNA in the presence of Cohex. It gave approximate values for the tension present when DNA collapses. The second article provided another theory as to why the DNA would condense and the resolubize under increasing amounts of Cohex. It suggests that as the concentration increases, not all of the Cl- leaves the Cohex, so there are increasing concentrations of Cohex+2 instead of the normal +3. This would mean that the Cohex is actually out-competing itself, causing the DNA to return to solution. It is possible that our results could be supporting this theory, although it is hard to tell without the Cl- concentration. This is unfortunately impossible to get because the solution was flooded with NaCl to resolubize the DNA. In the afternoon, the nitrogen finally got here, and I was able to resume testing. The first thing that I tested was the fourth of four initial runs that I did on the real sample.