Author: Prof. Andresen

On Thursday, we continued our DNA production process. Our main goal Thursday was to conduct a trial digestion of a small sample of DNA to find the right amount of time to let it digest for. This essentially ruined the sample of DNA, but was necessary so we know how long to digest the rest. To do this, we split a small amount up into ten samples, and put in micrococcal nuclease. Then we let the samples digest, and stopped them in 5 minute intervals. When we analyze these samples, they should give us a good idea of how long to digest the rest. In the afternoon, I did some more research and worked on my presentation for Friday.

On Friday, we had our group meeting, and everyone gave presentations on their research. This went pretty well, but it took the majority of the morning. Because we had put in too many hours earlier in the week, we took the rest of the day off.

Yesterday we continued the filtration of our DNA sample with additional centrifugation and stirring. We then proceeded to change the buffer of the solution by placing it in a dialysis bag with a different solution surrounding it overnight. I then ran a final test on my calibration of the spectrometer, in preparation to start testing on the actual samples today or tomorrow. I prepared three solutions of known concentration, and the results were fairly accurate, which is good. There seemed to be something wrong with the axial readings, because they were all negative, with very little intensity. We’re going to do some maintenance on the spectrometer today and that should fix everything up. I also continued reading the electrostatics paper, and with Professor Andresen’s help it began to make sense. I now know a bit more about the actual physics behind the competition of ions to be attracted to DNA, which is exactly what my tests will be studying as well.

Today we finished our last rinse and spin to get our the rest of the floating junk from our DNA samples. After we pi-petted out the clear leftovers we then dropped them in a dialysis tube in a Tris, Sodium, EDTA bath and left the chromatin dialyzes over night. I then continued reading my paper on Chromatin for my presentation Monday.

Today we worked on obtaining actual DNA from our red blood cell samples. This process consists of several repetitions of filtering and putting the solutions in the centrifuge. This also required the use of several different solutions that we prepared, including Tris/HCl, PMSF, EDTA and micrococcal nuclease. After essentially causing the red blood cells to explode, we used the micrococcal nuclease to eat away at the remaining substance. We controlled this reaction by controlling the temperature (putting it on ice) and by introducing EDTA to remove the calcium from solution.

Today we started the long process of separating out the nucleosomes from the red blood cells. we began by washing and centrifuging the red blood cells 4 times until basically the red had separated and we were left with a semi snotty mixture. we then cleaned the snotty nucleosomes again and collected the final solutions and combined them. We then added a mixture that broke up the nucleosomes and made them less gooey and then centrifuged again and took the liquid that was left over and tested the absorption of the leftovers and got a strange step graph where 260 nm was the middle stem. We are currently adding a salt mixture and about to do more centrifuging

Yesterday I started the day by analyzing my results from the previous day. These results were by far the most accurate, but there were still a few issues. For some reason, when I ran the calibration series as a sample, they all were read very accurately, all less than 2% error. But for some reason my other series was still 4-8 % off. This leads me to believe that this solution wasn’t prepared correctly, because the spectrometer is clearly calibrated correctly now. It would seem that spacing the calibration samples linearly and using a non-linear yields the best calibration. Later this week I will carefully prepare a new sample series just to confirm this. I also began reading another electrostatics paper that I will try and present on Friday.

Today I tried the forcing the calibration line through zero for the data set I have been working with, and I found that it was no different than the weighted linear fit. I also tried constructing an actual concentration versus reported intensity graph myself, and seeing how to best fit that curve. I found that a polynomial fit worked the best, and my result was exactly what the computer had done for its fit. Next, I designed and prepared a new calibration series, one that is equally spread over the range of concentrations we are working with. We also made a few changes to the testing method the machine uses, changing the amounts of time it is permitted to read each sample. I think that these two changes should help eliminate a lot of the systematic error we have been seeing.

Yestereday I investigated more into how the machine calibrates itself. I read all of the help files on the different types of calibrations, and tried three different methods on my latest set of data. These methods were linear, weighted linear, and non-linear. I found that weighted linear and non-linear were more accurate than linear, but neither provided the accuracy that we really need. Today I’m going to try another type of fit that forces the calibration line through 0. I am also going to try plotting a graph of concentration versus intensity on my own, to see how the calibration process works. I’m also going to make another calibration series that is equally spread over the range we will be working in, instead of the logarithmic set we have been using.

Today I began by combining 3 nucleosome samples together and using the spectrometer to see how good the sample was. Unfortunately it wasn’t a good sample and I kept getting this weird peak in my data at 300nm and then had a slow decline to 220nm. So we skipped those nucleosomes and I tested two more samples that we had. The first had the same curve and the second had the curve we wanted but it was already diluted too much to be of any use to us. After that i tested the last two nucleosome samples that were in the freezer and the first one had kind of the curve we wanted but it peak was from 280-240 nm. The last sample had exactly the peak we wanted but the professor had no idea what was in it. He decided we would go over to the biology department and test the DNA to see how many base pairs it had but we found out we were missing a chemical we needed to do the test so we have to wait. For the rest of the day i spent time gathering supplies and reading up on a similar experiment to the one I’m doing.

Today I took a break from experimenting after yesterdays hectic day. I instead did some reading especially on the diffusion technique I am going to use tomorrow with the old nucleosome samples that we have in the freezer. I am going to use the Mg samples that I made a few weeks ago and run them through the nucleosomes in a centrifuge and measure the amount that is absorbed by the nucleosomes.