Author: Prof. Andresen

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.

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.

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.

Well, this was an exciting day. It started out with the arrival of the full lab. Travis and John began working at 9am this morning and quickly got busy reading for their various projects while I worked on preparing samples. (For the record, Travis was given the Nucleosome Packing project while John has been assigned to the DNA Liquid Crystal experiment.) I had a heck of a time getting the calibration solutions prepared as when I was ordering I neglected to buy a funnel for pouring solutions from a centrifuge tube to a volumetric flask. A little bit of begging over at chemistry and the help of Prof. Shelli Frey and I finished the job just a few minutes into lunch.

It was important that I finished these calibration solutions as they were needed for the afternoon competition. Basically, the idea was that Travis and John would make five solutions using their best pipetting skills. This activity had two purposes: One was to give Travis and John a chance to practice said skills. Pipetting is something that is quite easy to do but takes some skill to do right. The second and more important purpose was to see how well we could all pipette solutions and how well the machine could measure the concentrations of the various solutions.

Here are the results:

Where the highlighted values are the predicted values. Everyone did pretty well. Travis knew he had a bit of an issue when all of his volumes were a bit high, but look at that precision. John seemed right on for the most part. What concerns me more, however is how poorly even the John and my values match the predicted value.

One issue is that these samples were originally meant to be diluted by 10x. We’ll try that tomorrow. I’m also planning on changing the pump tubes out and trying to take more replicates. We eventually want an accuracy of ~1%.