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Note: I realize these posts may be confusing to those outside of biology/biochemistry fields. In general, gel electrophoresis separates molecules by size: In this case, biggest are at the top and smallest are at the bottom. For more, look at this great animated introduction.

As you remember from my last post, our last gel ran quite well, but the results showed that we had failed to digest our inter-nucleosome DNA. This time, we modified our digestion process (and by modify, I mean went back to the process I had been using before I decided to “improve” my process).

This time however, we have another issue. The gel itself gave us problems. There were a couple issues layered on top of one another, but the main one was that our power supply that runs the gel decided to quit some time in the middle of the night. This had two effects, one was that the DNA had time to diffuse and migrate in any old direction it wanted to during the time that the power supply was off. This causes a broadening of the bands which I think we are seeing here. Another issue was that it forced us, due to time considerations, to ramp up the voltage to very high values which I think is what caused the intense upside-down V-like shapes to the bottom of our gels. Finally, we ran the gel a little too long losing our 100bp marker. In the previous post, you could see the 100bp and 330bp markers, but I am pretty sure that the marker you see in the first lane is simply the 330bp marker. This makes it impossible to determine as, even though some of the middle markers are resolved, we have no way of counting up from the markers (each sub marker is 10bp).

As for the digestion itself, things look pretty good. We got rid of our smear of DNA and it is obvious that the DNA is migrating to approximately the correct place. There are only two issues I am worried about. One is that aside from the zero time point (the second lane), most of the lanes look the same. In other words, it seems as if we are completely digested by 5 minutes.

The second issue is that the lanes that do look different are completely random. The corresponding times are 20, 40, and 50 minutes. By 50 minutes, we shouldn’t see anything above that band of brightness on the bottom. The fact that it is random seems to indicate a possible pipetting error. However, I ran and loaded two separate gels, so the pipetting error would have had to have come earlier. We’ll try going over our process to make sure that this isn’t an issue.

Other than that, it’s back to do the exact same thing again. This time we’ll be using a different power supply so we don’t run into the same problems.

Yesterday I began analyzing my results from the previous day’s testing. The results seem to show a clear pattern, for as more Mg is added to the initial solution, more Mg is precipitated, suggesting it out competes the Cohex. In general though, as more Mg was added, less DNA is found in solution, suggesting that Mg does not precipitate the DNA nearly as well. I also began working on a report that shows my process and an overview of my results so far. I also prepared another set of samples that should be exactly the same as the ones I ran the last few days. Running these samples again should give me a data set large enough to get some good statistics for my results.

Today I spent the day making new timed digestion samples that we could run in the DNA gels. After I finished the final bath that the samples take in 50 celcius I created 10X solutions with each of the time trials because last time we made a gel it was very bright which is an indication that there is too much DNA. We ran the DNA gels overnight and tomorrow we are going to dye them and see our results

I finally have real, tangible, reasonable results from our collaborator’s samples! I spent the morning making new calibration standards and a new set of test samples. I also started reading an article about questionably-plausible applications of this research in gene therapy. It also has applications in designing safer and more effective drugs, as well as learning how viruses pack DNA so efficiently, which would help us fight them. Anyway, the results look fairly consistent at least, I would say accurate but I’m not sure we know what they’re supposed to look like. There may be one outlying point that I need to check into, it’s possible that I messed up the preparation. The results seem to be telling me things, I’m just not sure what those things are yet. It is clear from the results that as you go up the series of increasing initial magnesium concentrations, the final results are that the magnesium out-competes the cohex. The unknown part is how and why this happens.

Today we got back our DNA gel and were surprised to find that each of the trials seemed to be very similar even the zero digestion sample. After a lot of thought the professor determined that the process we did for digesting the nucleosomes earlier was around 100 times off than the recommended so that our samples were not originally digested all the way that we wanted. Tomorrow I am going to run a timed digestion again except this time we are going use more nuclease in order to make up for the digestion that we haven’t done yet.

As Travis mentioned in an earlier post, today we finished running our gel. With the help of the Steve James lab, we imaged our DNA. The results were not promising.

What you see on the left is just the DNA ladder. Those numbers correspond to the number of base pairs (bp). On the right, in the lane labeled 1, you see our DNA ladder with red lines tracing the corresponding markers. As you can see, we can clearly see the 330bp and 100bp markers. What we can’t see are all of those nice little lines in between. This could be a problem in the future, but is small beans compared to the mess further to the right.

In lanes 2-12, we had placed DNA samples that had gone through various times of digestion. What we were trying to do was take DNA that was all kinds of different lengths and snip most of it down to 146bp. Each lane corresponds to 5 minutes later than the one before it. So lane 2 is time zero, lane 3 is 5 minutes of digestion, lane 4 is 10 minutes of digestion, and so on. Now, you don’t have to be an expert to realize that lane 2 looks identical to lane 12! This is a bad sign. But what does it mean?

There could be many issues: The gel could have been run incorrectly, the samples could be contaminated, or we could have so much DNA (gels aren’t normally this bright) that it somehow makes us unable to see what is actually happening. But I’m pretty sure none of these are the cause.

The procedure for digestion I used was a modification to the procedure I had used the first time I did a nucleosome preparation. The modification required a few more measurements, but it was claimed in the paper I pulled it from that this procedure gave better results (i.e. more nucleosomes at the end). We did these extra measurements (I believe correctly) and added the amount of nuclease (the digestors or eaters of the DNA) that the new procedure called for. However, as both Travis and I found out in later calculations (that perhaps should have been done before the trial digestion) this new procedure called for 100 times less nuclease than the former procedure called for. The final result: No digestion.

Luckily enough a) We were doing a trial digestion, not the real thing, so we didn’t lose any real amount of samples and b) It is quite easy to repeat the trial digestion with the old method and then rerun our gel. And that’s exactly what we are going to do tomorrow.

Wish us luck.

This morning, I analyzed the results from my first tests on the real samples. Unfortunately, the machine was not properly calibrated to work well at such low concentrations. The pattern we were expecting to see was for the magnesium concentrations to increase across the series, and for the cobalt and phosphorus concentrations to decrease. The data slightly resembled these trends, but it was also riddled with confusing negative values. These negative values are what suggest that the machine is not calibrated properly. After lunch, I set out to see if the machine could be calibrated to be accurate at very small concentrations. I diluted my previous calibration series X100, because that is more around the range we are dealing with. This is because we diluted the samples X100 to try and conserve the sample if possible. I ran the tests this afternoon, and from my first look, it appears that the machine works quite well at the single parts per billion range, which is impressive. I should be able to start getting realistic data on the real samples tomorrow. One problem that I did have yesterday was that as I was running the real samples, the plasma torch was burning bright orange, and it seemed much stronger than normal. This is disturbing both because it could be damaging the machine, and it will almost definitely throw the results off slightly. I also got a strange error about mercury lines this morning, but it seems everything is alright.

Today we ran our DNA samples that had been eaten with the nuclease and then stopped with the EDTA through a DNA gel which measure how many base pairs are present in the DNA. It does the by running a current through the gel and the very negative DNA moves towards the positive end of the current. The smaller the base pair number the faster the DNA moves so the higher base pairs would be at the top of the gel and the lower at the bottom. Because it was running so slow we turned the voltage down and allowed the gel to run overnight, and tomorrow we are going to dye the gel and look at it with a UV light.

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.