Andresen Lab Blog

Well this has been a great week. Here’s what we accomplished:

1. Travis ran a Mg concentrations series on some nucleosome samples. He, for the first time in Andresen lab history, saw the condensation and precipitation of nucleosomes. I don’t have his data right now, but perhaps he can upload it and explain what happened. Next week he will be running the same experiment, only this time using equilibrium dialysis. This is exciting as this is the method we will eventually use for our nucleosome experiments.

2. John is really honing in on our uncertainties in our ICP-OES measurements. He’s run somewhere around 30 samples total so that we have been slowly able to eliminate one possible cause after another. The last batch of samples he made before going home this afternoon should either confirm or eliminate our final couple of theories on the source of our ~5% systematic error. If he eliminates all of our explanations, then I guess it’s back to the drawing board.

3. I’ve gotten the OK to collect chicken blood. Around 10am this Tuesday, I should be in Waynesboro, face-to-face with a pretty unhappy chicken.

And that’s all for this week. Couldn’t have asked for a better one. (Except for that darn UV detector that still hasn’t shipped from Fisher…)

Many of you might have wondered in the past: Where does your lab get samples from? You probably assume (correctly) that the normal procedure is to find a catalog, look up the thing you want to buy (for instance, DNA), and order it. A second method is to grow your sample, normally in bacteria, to get large amounts of sample.

For our nucleosome experiment, neither of these are practical or economical for the amounts that our group will use. So instead, we need to get nucleosomes from a source that has already done the work of making them. The preferred sources are from calf thymus (the thymus is an organ in front of the heart that is a vital part of the immune system) or chicken erythrocytes (chicken blood). I prefer the chicken blood as it is something easily collected, handled, and processed. (As an interesting side note, the blood could come from a non-mammal source. Mammal red blood cells do not have nuclei and therefore do not have significant quantities of DNA.)

Now I like to have approximately 200ml of chicken blood to create my samples. One option is to extract 1ml of blood from 200 chickens. With each chicken containing approximately 100ml of blood, they probably would barely know it. However, because it is much quicker (and as these chickens are going to be killed anyway), I prefer to collect all 100ml from each chicken.

This requires me to find a chicken butcher that allows me to collect blood from the slaughter. Normal slaughterhouses, with their incredible efficiency (for some disturbing pictures, try here) are not a viable option. So I need to find a small scale farmer that hand slaughters his or her chickens.

I was first turned on to Rettland Farms, a wonderful local farm that sells free range (really free range, unlike most labeled as such) chickens. He pointed me to his processor over in Waynesboro, PA. To make a long story short, the processor is doing relief work in Haiti, will be out of the country until August, and has someone else filling in for him. I put in a call to the cellphone of his replacement slaughterer (pictured at right), a member of a Mennonite farm collective.

So to wrap it all up, I’m waiting for a Mennonite slaughterer to check his cell phone’s voice mail and return my call so I can ask him if I can hold a beaker under his chickens as he ends their lives.

Somehow we didn’t cover this part of science in grad school.

This morning, I analyzed the results from my tests yesterday, and I found some strange results. In general, all of the predicted solution concentrations I got from the spectrometer read-outs were at least 3% larger than the concentration to which I tried to prepare the solutions. In fact, all but one set of data was over the prepared concentration. This is rather a rather disturbing systematic error, and the sources need to be identified. Though the results are not as accurate as we need, the good news is they are very precise, because if you ignore their consistant 3-6% overreading, they are then within 1%. These could be all random errors, but that seems unlikely because of their consistency. They could also being coming from my technique of solution. It could also be a systematic error coming from the pipettes. It could also be an issue with the calibration with the machine. I will investigate the source of the error more tomorrow. I did a few tests at the end of the day, and they showed that the pipettes might be cause a small portion of the error, but nowhere near all of it. I also did more research into the cobalthexamine project that I will be working on throughout the summer.

Today I increased the amount of MgCl I added to the solution in order the speed up my result process. For a few trials my solutions stayed at or below the .1 concentration mark but as time progressed my concentrations began to increase at around 48 micro liters of MgCl being added to the DNA solution. This meant that the nucleosomes that had clumped together earlier and fallen to the bottom of the solution were breaking apart and floating back up to the top. I never got my results up to a definite high concentration because as I kept taking data the Professor noticed that the amount of absorption at the 280 nm mark was very high and getting higher which according to him meant that there were excess proteins in the solution which could be the result of bacteria growing in the solution. I think the problem might have been that I started out with very small amounts at the beginning which slowed my result process and caused it to be a lengthy experiment. Also the solution that i was using were leftovers and might have just been too old. I hope to try and redo this experiment after we create our new nucleosome solutions in the following weeks and plan to use my results from this trial to influence how I go about taking data with the next experiment.

I spent the first half of the day today organizing and analyzing the results from yesterday’s samples. The results I received for the predicted concentrations of my soultions were pretty consistently 5% away from the values that I expected. This may be a problem with my preparation of the solutions, or it could be an issue with the calibration or conditions of the machine. In particular, the axial readings for the Magnesium concentrations were consistently 30%+ away from the expected value. For the most part, these results should be ignored because they are oversampled. Also, I found that consistently the first sample I tried to run was not drawn from the test tube, which led to a lot of strange negative results for one of the samples.

To try an look further into these issues, I prepared and tested a large new set of samples. This new set has two magnesium series, a cobalt series, and a phosphorus series. This should help identify where some of the 5% error is coming from. If it is a problem with the machine or the range of concentrations we are running, more data will make this easier to see. If it is a problem with my preparation, this will also become obvious. I ran the tests at the end of the day yesterday, and will analyze the data tomorrow.

Also, we are looking at changing the calibration method for the tests, because of the strange magnesium readings we were getting for the axial view. It is possible to use a non-linear fit for the calibrations, and we will see if this helps tomorrow.

Today was a continuation of yesterdays research I continued to add MgCl to my DNA solution and test the amount of absorption of the light we put through it. After hours of taking data i finally got to the bottom of the curve where the MgCl has pulled most of the nucleosomes to the bottom of the solution and have just started to get results of the nucleosomes freeing themselves and returning to the top of the solution. I will continue to add larger amounts of MgCl to the solution tomorrow and hopefully get the data that I am looking for.

More progress today, although not much on my part. I’ll let John and Travis talk about any major results they found.

I’ll tell you one thing, they’ve been working hard. I’m getting three times as much work done as I usually do. At this rate, we’ll have all of science figured out by the end of the summer.

I, on the other hand, got a minimal amount of work done. I worked on getting Matlab installed, which is what we will use to analyze any x-ray data we end up getting. Although recent communications with the head of the beamline we were planning on using seem to suggest that this work will be later than we had hoped. Matlab can also be used to make some nicer plots of any data we want to publish. A little bit of friendly advice: Friends don’t let friends publish excel graphs.

John and Travis will be happy to know that I ordered a bunch (25) of tube holders so that we aren’t fighting each other for them as well as a bunch of other small things needed in the lab.

As for tomorrow, Travis should be finishing up his experiment today or early tomorrow. We’ll probably pause on that so he can write up his talk that he’s going to be giving on Friday. The same thing goes for John’s current project. Hopefully after a day of thinking and looking at data on Thursday, we’ll be going full steam ahead again on Friday.

Today in the morning, I read through an article titled “DNA Aggregation Induced by Polyamines and Cobalthexamine”. This article discussed how spermidine, spermine, and cobalthexamine could cause DNA molecules to precipitate out of solution. Then, further addition of these cations can also cause the DNA to become soluble again. The concentrations required to make the DNA precipitate are basically independent of how much DNA is in the solution. This precipitation is not caused by a classical “salting out” situation, where high salt concentrations change the activity of the water. One explanation for the precipitation and then resolubilization is that it is caused by short-range electrostatic attractions. Another explanation is that the DNA undergoes a charge reversal, but this explanation is less likely.

The rest of my day was spent preparing three different series of test solutions to test the calibration of the spectrometer. The reason for these test solutions is to find the optimal reading conditions for a later test of a specific sample that has a similar make up. I made a Mg series that was varied over 7 concentrations, a Co series over 4 concentrations, and a P series over 3 concentrations. These will be used to see how accurately the machine can read my “standardized” samples, but also I guess it will measure how well I can make solutions. At the end of the day, I was just finishing the tests, and did get to finish looking at the results. What I did see did seem to have a systematic difference of around 5%.

Today after a bit of reading on a nucleosome project I began measuring the amount of absorption of different wavelengths as the were shown through the DNA sample. For every trial i would add a small amount of MgCl to the solution and mix it up. As the amount increased there were white clouds that appeared which were the nucleosomes clumping together and falling to the bottom. My results didn’t show what we were looking for and after a bit of thought the Professor determined that we needed to take a few more trials before we got the results we wanted. Tomorrow I will continue adding more MgCl to the solution and hopefully will get to the point where most of the DNA clumps to the bottom and the amount of absorption decreases and then as more MgCl is added the DNA will somehow unclump and reappear at the top of the solution.

Well, we certainly had a productive day today.

To start things off, Travis started a new sub-project for the nucleosome experiments. He is looking at how the nucleosomes I made respond to an increasing amount of magnesium (a +2 ion). Theory says that as we add more magnesium the nucleosomes clump up and fall out of solution. This is an indication that they are attracting each other so strongly that they would rather be next to each other than dissolved in solution. The crazy thing about nucleosomes (and as it turns out naked DNA does this as well in different solution conditions) is that if you keep adding more magnesium, the nucleosomes go back into solution (in other words they no longer attract each other). This is pretty crazy if you think about it: You make the nucleosomes attractive by adding +2 ions and then get them to then be repulsive by adding more +2 ions. This is one of the reasons this system is so interesting to look at.

Travis started this process, slowly adding more and more +2 ions. And low and behold, we saw some aggregation happening (in the form of white wisps of material in our solution). Unfortunately, they redissolved after a bit of time.  Tomorrow we’ll try to get up to higher concentrations of Mg and hopefully see the full transition.

John, meanwhile, has extended yesterday’s experiments to look at a lot of samples (11 or so, I think). The reason for all of this is that we have some samples from our collaborator that we need to run. Unfortunately, when we run them, we destroy them, so we need to do it right on the first shot. To ensure that we are taking the measurements under the correct conditions, we are going to look at a lot of samples that are very similar to the ones we want to look at. Once we get the correct conditions for those samples and are really happy with how they are running, we can think about looking at the real samples. Not the most exciting thing in the world, but that’s science folks: There’s always a lot more build up to the experiments than there is time spent doing the experiment itself.

Finally, Xiangyun Qiu  and Chongli Yuan are joining me in a collaboration to look further into the mechanisms behind nucleosome folding. We are hoping to do and x-ray run later in the year to look at what happens to nucleosomes when you remove or modify the tails or put them in different types of solution. Our preferred tool, SAXS, should give us some idea of what’s going on.