Author: Prof. Andresen

I spent most of today analyzing and writing up my results from the past few days. Some good news, is that the data sets are fairly consistent with each other. I graphed the charge that was contributed by both ions to compensating for the DNAs negative charge, and it actually appears to have a somewhat linear relationship. There are two points that do not fit the linear fit, the 8th point and the 10th. The 8th is somewhat disturbing, because it appears to be random. I originally thought I might have prepared the solution poorly, but now that I have run another differently prepared solution, I find that hard to believe. Who knows though, it is very possible it should be like that. The graph on the left is the average of the 3 data series that I have collected so far. It is also a little strange that there are often more than 1 total charge contributed by the sum of the Mg and Cohex, because DNA only has 1 negative charge per phosphate. Because the Mg and Cohex are normalized with the P concentration, there should never be more that 1 charge total. This suggests either some error, or some sort of overcharging. I am nearly done with the analysis paper I have been writing, and will most likely post it on here when it is finished. Next week I will be finishing analyzing this data, preparing the new calibration standard, and running as much more sample as possible.

Thursday was a pretty relaxed day as we waited for our DNA gels that shutdown over night to finish up. While we waited I read an paper on how Magnesium affects chromatin but it was a little dense and hard to understand so I’m still making my way through it. When we finally got our DNA gels and pictures of them we found that we had let them run too long and that the data we were looking for had run off the gels. But we had better looking gel than our first try so we are narrowing down our results and should get it the next time. Today I re-tested the old nucleosome samples to determine which one that we had was the best sample compared to our new DNA. after some confusion with the results I finally got the data and determined how much nucleosomes per gram are in the solution and how much we need in order to have a good comparison with our new DNA for the next time we run a gel.

Yesterday I began analyzing my results from the previous day’s testing. The results seem to show a clear pattern, for as more Mg is added to the initial solution, more Mg is precipitated, suggesting it out competes the Cohex. In general though, as more Mg was added, less DNA is found in solution, suggesting that Mg does not precipitate the DNA nearly as well. I also began working on a report that shows my process and an overview of my results so far. I also prepared another set of samples that should be exactly the same as the ones I ran the last few days. Running these samples again should give me a data set large enough to get some good statistics for my results.

Today I spent the day making new timed digestion samples that we could run in the DNA gels. After I finished the final bath that the samples take in 50 celcius I created 10X solutions with each of the time trials because last time we made a gel it was very bright which is an indication that there is too much DNA. We ran the DNA gels overnight and tomorrow we are going to dye them and see our results

I finally have real, tangible, reasonable results from our collaborator’s samples! I spent the morning making new calibration standards and a new set of test samples. I also started reading an article about questionably-plausible applications of this research in gene therapy. It also has applications in designing safer and more effective drugs, as well as learning how viruses pack DNA so efficiently, which would help us fight them. Anyway, the results look fairly consistent at least, I would say accurate but I’m not sure we know what they’re supposed to look like. There may be one outlying point that I need to check into, it’s possible that I messed up the preparation. The results seem to be telling me things, I’m just not sure what those things are yet. It is clear from the results that as you go up the series of increasing initial magnesium concentrations, the final results are that the magnesium out-competes the cohex. The unknown part is how and why this happens.

Today we got back our DNA gel and were surprised to find that each of the trials seemed to be very similar even the zero digestion sample. After a lot of thought the professor determined that the process we did for digesting the nucleosomes earlier was around 100 times off than the recommended so that our samples were not originally digested all the way that we wanted. Tomorrow I am going to run a timed digestion again except this time we are going use more nuclease in order to make up for the digestion that we haven’t done yet.

This morning, I analyzed the results from my first tests on the real samples. Unfortunately, the machine was not properly calibrated to work well at such low concentrations. The pattern we were expecting to see was for the magnesium concentrations to increase across the series, and for the cobalt and phosphorus concentrations to decrease. The data slightly resembled these trends, but it was also riddled with confusing negative values. These negative values are what suggest that the machine is not calibrated properly. After lunch, I set out to see if the machine could be calibrated to be accurate at very small concentrations. I diluted my previous calibration series X100, because that is more around the range we are dealing with. This is because we diluted the samples X100 to try and conserve the sample if possible. I ran the tests this afternoon, and from my first look, it appears that the machine works quite well at the single parts per billion range, which is impressive. I should be able to start getting realistic data on the real samples tomorrow. One problem that I did have yesterday was that as I was running the real samples, the plasma torch was burning bright orange, and it seemed much stronger than normal. This is disturbing both because it could be damaging the machine, and it will almost definitely throw the results off slightly. I also got a strange error about mercury lines this morning, but it seems everything is alright.

Today we ran our DNA samples that had been eaten with the nuclease and then stopped with the EDTA through a DNA gel which measure how many base pairs are present in the DNA. It does the by running a current through the gel and the very negative DNA moves towards the positive end of the current. The smaller the base pair number the faster the DNA moves so the higher base pairs would be at the top of the gel and the lower at the bottom. Because it was running so slow we turned the voltage down and allowed the gel to run overnight, and tomorrow we are going to dye the gel and look at it with a UV light.

Today I continued my research into the Poisson-Boltzmann equation, as well as into an experiment done previously by Professor Andresen. His experiment used X-ray scattering instead of an OES, but I found one of his results to be very interesting and relevant to the experiments that we are doing now. He found that inter-DNA attractions began somewhere in between 1 ion per 5 base pairs and 1 ion per 4 base pairs. This is particularly interesting because the inter-DNA forces are what we are investigating, and our results should bare some resemblance to these numbers. In the afternoon, I began running the actual samples from our collaborator. I started with the first series, which is 1mM Co and ranges from 0mM Mg to 22.5 mMMg. I used a 100X dilution of these starting values though. I will analyze the results that I got tomorrow. I think that the concentrations might be a bit too low for accurate results, but they should give us a good first impression of what to expect.

On Thursday we took our final nucleosome solution and mixed a small amount of it with nuclease which ate the DNA that was between the nucleosomes. We put this mixture into a 37 degree Celsius bath and at 5 minute intervals took out a small portion and put it into a separate test tube that we then added EDTA to in order to stop all protein from working. This made sure that the nuclease would stop eating at our nucleosomes. We did this in order to determine how long we need to allow nuclease to eat at our nucleosomes to get the most single nucleosomes that we can. On Monday we are going to see how many base pairs we have in each of our timed solutions through DNA gels which when compared to a known amount of base pairs will tell us which solutions are best.