Author: Prof. Andresen

Today I made and ran a second series of concentrated samples for the first series. The results came out somewhere in between the diluted series and the first concentrated series that I ran. This is promising, because the results should all be the same. It is possible that there is some sort of concentration dependent interference that is happening, which could be causing some of the differences. Today, I decided to start running concentrated trials of the second series, and started preparing the samples. This should be easier because there is much more of the second series left. Overall, I am finding that the eighth and tenth points are still uncharacteristically high, which leaves me to believe it is something in the actual sample preparation. This is one of the only explanations I can see left, because I have ruled out most of the possible problems on the testing end. One possible explanation is that not all of the Mg buffer solution was removed when the DNA precipitated.

Today, I wrote the second of two results reports, and updated all the graphs for the second series. It was Matt and Andrew’s last days, so we had their final presentations today as well.

On Thursday, I began by analyzing the concentrated trial for the first series. It came out to be significantly lower than the eight diluted trials I had run previously. By lower, I mean the the normalized concentrations for the concentrated trial should have been the same as the diluted trials, but they were consistently below the diluted trials. This was kind of disturbing, and has prompted me to run more concentrated trials. I also updated my results report, and fixed and updated all of the graphs that I have been creating. I also consolidated and organized all of my data and graphs.

Yesterday, I started by running the last trial of the second series. I also prepared a few more tests and ran them as well. The first was a new test for chlorine, which worked slightly better than the first. Part of the problem was that I didn’t prepare the NaCl standard quite right, but some of the results still come out negative. I also ran some of the Chromatin that the professor and Travis have been working on, and found that it had higher calcium concentrations than they expected. I also designed and ran a highly concentrated series of the first series. This should help identify what is actually going on in the last few samples of each trial. They have been fairly random, but almost always are way above where I would think they should be. This is attributable to the fact that are very low concentrations of all of the elements. This makes sense, because less DNA is precipitating.

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.