August 5th

Today I put the finishing touches on my presentation. I added a few graphs from the first series, and changed around a bunch of the slides. Other than that I just worked on an outline for my own benefit. After listening to Alex's presentation, I gave mine and received criticism on it. I still have some work to do on it, but it should be ready for my presentation in the spring.

August 4th

Wednesday, I spent pretty much the entire day working on my presentation. I have made a series of animated pictures that will help explain overcharging and the counter ion density wave theories. I also recruited the aid of Atwater Animations Inc. to help with some of the method slides. I think that over all, my presentation is really coming together.

August 3rd

On Tuesday, I finished up with my final analysis of the data for the second series. I calculated all of the averages a different way, one which I found to be a much better representation of the data. I got the concentrated series to agree very closely with the diluted trials, which is very promising. I also began updating my presentation for Thursday.

August 2nd

I spent most of Monday organizing and analyzing the data from the second series. This included looking through all of the raw data from all of the trials, and making sure there were no large problems with it. I also updated it to take into account the standard deviation of the mean.

August 3

This is the website John and I created
http://gburgsummerphysics.webs.com/

July 29

Today I ran 2 digestions with the only difference being how much nuclease i put in each sample. In one i put a 10x dilution of our nuclease and in the other i put a 100x dilution. I kept everything else the same and used our old protocols to complete the digestion. On Monday we are going to run the gel because we have been kicked out of the chemistry department for the rest of the week.

July 29th

On Thursday, I made some new spreadsheets to analyze the data in a different way. I am trying to average the data in different ways, by averaging the values across the data series first. I think that doing this way is more accurate, because the trials are fairly consistent, and this helps to take into account where most of the error is coming from. This process seems to be making the data agree with itself more, which is promising.

July 28th

On Wednesday, I finished running the concentrated trials of the second series. I also reprocessed a few of the earlier trials, trying different calibrations. I discovered and correct one major problem, which was that I had run out of sample in one of the trials. This was throwing that trial way off, because it was taking values when there was no sample left. Reprocessing some of the data with different calibrations also helped make the data more consistent.

July 27th

On Tuesday, I continued to prepare and run the concentrated trials for the second series. So far things are going well, but the data is not quite matching up with the diluted trials. This could be because of the way I am analyzing the data, or also from problems and inconsistencies in the data itself.

Many general lab updates

For our nucleosome project, we have had some setbacks, most of which we have recovered from. As you remember from last time, our column had broken. That is still the case and we are still waiting for a replacement as it is on back-order. What I had failed to mention was that the sample itself had also had some issues. Namely, there was a white powder that had precipitated out of the sample and to the bottom of the tube. This was disturbing as I was relatively sure that this was all of my nucleosomes aggregating and falling to the bottom of my tube (something they are only supposed to do when I want them to). Since we were stuck without column, I thought I would investigate this issue.

It turns out that nearly all of my nucleosomes had aggregated. After some investigation, it turns out that the nucleosome sample was actually in 10mM Calcium Chloride (a +2 ion) instead of 1mM. This is an issue because nucleosomes aggregate at about 5mM of +2 ions. It is also a problem because the EDTA we add to stop the digestion is added to stop 1mM of CaCl2, not 10mM.

The first thing I needed to do was get the nucleosomes back in solution. We did this by dialyzing the nucleosome solution (and the precipitate) with 150mM NaCl (a +1 ion). This is done by putting the nucleosome solution in a bag that only lets ions and water pass through it, but not the nucleosomes. It turns out, if you add enough +1 ions, they will displace the +2 ions at which point the nucleosome should go back into solution. After a day of dialysis, we had significantly increased the amount of nucleosomes in our solution. After a weekend of dialysis, we had gotten back basically all of our nucleosomes. Now we had to make sure that they had been digested correctly (especially checking for over-digestion which shows up as smearing on our gel).

The results look like this. Now the furthest column to the right is our DNA ladder. This is used to let us know how long our DNA is. You match the known length of the ladder band to your sample. The bright band at the bottom is a 100 base pair DNA. Each step "up the ladder" is a 10 base pair step (110 base pair, 120 base pair, etc). The lane immediately to the left of the ladder is our sample. If you look and count carefully, you can see that it lies between the 140 base pair and 150 base pair "steps" of the ladder. This is perfect as the DNA should be 146 base pairs! Even better, there is no "smearing" to lower parts of the gel, which would indicate over digestion. You can see an example of this in a different sample that was run in the lane immediately to the left of our sample.

So as a wrap-up, we have the same amount of nucleosome we started with, all in solution and digested to the correct length. All that is left to do is to separate out any small amounts of double or triple or larger nucleosome arrays and we will be done. Since we are still waiting for our column to arrive, we may end up doing this through a sucrose gradient instead.

Not to be shown up, John is busy plowing through the ton of data that he has taken. Now that we have all of the data, we need to figure out why some of it looks great and some of it looks not-so-great. Things looked pretty bad at the beginning of this process, but each time I check in with him, he seems to have found another correction to make the data fall in line. If all goes well, we'll have it all whipped into shape by the end of this week.

July 27

Today I ran tests on the Lambda 25 to see if it is properly working, and it passed all the tests I could do without having to make any samples. I also created an instruction sheet for the machine with the basics so that anyone can now use it.

July 26th

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.

July 23rd

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.

July 22nd

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.

July 21st

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.

July 20th

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.

July 21

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.

July 19th

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.

July 16th

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.

July 15th

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.