Andresen Lab Blog

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.

Well, we keep getting better at least. This digestion showed a much better digestion. As you can see, the 0 minute digestion showed a smear of DNA, while all lanes that were digested for 5 minutes or more showed a nice single band that can be believed to be around 146bp. As can be seen we have a few issues.

First, the digestion is supposed to take around 30 minutes. This is helpful because it means that you can make sure that you are just digesting to 146bp and not overdigesting. Ours is taking 5 minutes. In our next digestion, we’ll try decreasing the amount of nuclease (the thing that “eats” the DNA) by a factor of 10. Hopefully we’ll then see a little more of a progression and less of a step function.

The second issue is that the gel still doesn’t look great. The first issue is the ladder. We obviously didn’t run long enough: Our 100bp standard only went about 1/2-way down the gel. However, conditions were controlled enough the last time that we should be able to have a nearly-perfectly timed run.

The other issue with the gel is that the DNA in all but the ladder lane is quite diffuse. It would be much easier to read if they looked like lines, similar to the ladder in previous gels. My theory is that this is due to the sample not being dense enough when loaded and therefore diffusing before it starts running in the gel. An easy fix for this will be to increase the amount of sucrose before loading these samples.

Finally, a problem with the gels that I’m not showing you is that I messed up the loading of the other gel. This is actually Travis’s gel. Mine didn’t have a ladder! Well, I’ll have to do better next time.

Things with John’s project are going quite well as you can see by his posts. We should have nitrogen tomorrow which will allow him to run more samples. After that starts up, we should be on our way to having all of the DNA aggregation samples done by the end of next week if everything goes well.

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.