Andresen Lab Blog

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.

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.

I’ll let everyone reading this blog in on a little secret. A good portion of a researcher’s time is spent on menial tasks and tasks that look very similar to “doing nothing”. Today was one such day for me (as was much of the week). I spent most of my time ordering supplies, running around borrowing/stealing supplies from others, and planning our future projects. This last one was the most difficult, as my brain has just not been cooperating lately. People will say that this is related to the 1-month-old baby in my house, but I personally just think it’s just the consequence of a more-hectic-than-normal week. To at least a little justify my presence in the lab, I poured a very dangerous powder, PMSF, into isopropyl alcohol  to make a slightly less dangerous solution. (Less dangerous because it’s a lot harder to inhale a liquid than a dust.)

In real news, we are going to be doing two rather important things next week. First, we are making our nucleosomes. This is exciting, although perhaps a bit early. It’s early because it is questionable whether we will have everything we need ready by that time. However, in science it is often the case that you must go ahead with plans and hope for the best. If you wait until everything seems to be ready, you never get anything done. The good news is that our Fisher rep claims our UV detector is coming on Monday. With some luck and some help from Prof. Brandauer in Health Sciences, we should be able to get that up and running on Monday. Additionally, I’m hoping to make all of the stock solutions we need on Monday so that we aren’t running around trying to make those all last minute. Our collaborator, Xiangyun Qiu, is coming on Tuesday to see the prep and generally just to visit, so it this coming week should be busy.

The other exciting thing is that I’m expecting to start running the condensed DNA samples that John has been preparing the machine for. Although he’s still having a hard time nailing down that last 2-3% of systematic error, I think we’re pretty close to being at a place that we can run at. I’m guessing that come Friday or so, we’ll be in business. As Xiangyun is the one that gave us these samples, we’ll also take some time during his visit to talk to him about the samples and our general running procedure.

So it should be an exciting first week of “summer”.

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.