Author: Prof. Andresen

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.

On Friday I spent the day preparing another trial digestion this time using .1 micro liters of nuclease instead of 1 micro liter. Because our previous gels showed our digestion ending very quickly we decided that we were adding too much nuclease making the whole process end after around 5 minutes. So I did another 50 minute digestion and made a 10X dilution. I also did a digestion on the collaborators nucleosomes using the same method. For these samples we also added a lot more sucrose than before also in higher concentration. The professor hopes that this will make our gels much more readable then they have been.

Today, I spent pretty much all day making new samples to test. I was anticipating getting the new nitrogen tank today, which would let me run some of these samples, but it hasn’t come yet. I have two new calibration standards, which are both designed to span the exact range I’ll be working with. They are both the same thing, but prepared twice just to make sure that sample preparation doesn’t have anything to do with the results. I also prepared these calibration standards to be tested for accuracy as well, by separating them into additional tubes. I also diluted the DNA samples into 4 different sets, each of which are 100X dilutions. Today, the professor gave me a paper that has possible applications to our project. The paper suggest that some of the Cohex did not completely dissociate in the original solution, making some of them +2 ions. This would help explain why our results are significantly higher than the existing PB theory would suggest.

Yesterday, I worked on analyzing the data in a few more different ways. I separated the data so that it showed just the average Mg, Co and P concentration. This helped me to explain why two of the points don’t fit in my contributed charge graphs. This is a combined effect from the Mg and Co values being higher and the overall DNA (P) concentration was lower. I also started to design and make the next huge set of samples that I will be testing when we get more nitrogen.

Yesterday I spent most of the day reformatting the data that I have already collected. Instead of comparing the results across the different trials that I have run, I compared the results across the different collection methods the spectrometer uses. This gave me three more sets to compare, the Radial view and the two Axial views. This took a lot of manipulating the data, because everything I have done prior has been set up to compare different sets. Fortunately though, the end result was that I confirmed all three methods are equally valid. This means that I can keep using all three sets of data without fear of one of them being massively inaccurate.

Today we poured and ran our DNA gels.. the professor put in the concentrated samples and I put in our 10X dilution samples. To get the samples in the gel we had to take a pipette and slowly inject the solution into the wells in the DNA gels… After a while this gets difficult because you really have no room to mess up because if you go side to side you can mess up the wells and if you go to far down you can puncture the gel. But I finally got the hang of it and after some time we were able start our gel at 10V. The professor decided to use a power supply from the physics department because last time when we used the power supply that is in the lab it shut down during the night and messed up our results.

Today I ran another timed digestion going up to the usual 50 minutes. I created a 10x dilution of each sample and also made a sample of the old nucleosomes we had in the freezer by adding just enough to make the concentrations of the new nucleosomes samples and the old sample equal. tomorrow we are going to run the DNA gel which will probably run overnight.

Unfortunately, the spectrometer ran out of nitrogen over the long weekend and we won’t be able to replace it for a few days. This means I have to put off testing for a few days, but it also gives me time to look at the results that I have so far and plan the next set of tests. Today I worked on finding the uncertainties for all of my data and seeing how they compare to the theoretical Poisson-Boltzmann prediction. A few things bother me about the final fit that I came up with, which is seen above. First of all, PB theory would suggest that there should never be more than 1 total charge being compensated for, which there often is. Second, the eighth and tenth data points are disturbingly large. Third, the error bars for only the eighth and tenth points can be seen on the graph, meaning they are by far the largest errors. Fourth, the PB predicted curves shown on the graph as “Theoretical” had to be translated to fit that well on the graph. The Cohex curve had to be multiplied by a factor of 1.25, and the Mg curve had to be multiplied by a factor of 1.6. This means that my results are systematically higher than they “should” theoretically be. Also, I believe that once the new nitrogen comes and some maintenance is done to machine, combined with the new calibration standards I will make tomorrow, the results be more precise. This should help identify where the problems are coming from. Also, I should say that the third trial I ran, which is identical to the fourth I have yet to run, was prepared slightly differently and has already shown to be more accurate.