Andresen Lab Blog

Yestereday I investigated more into how the machine calibrates itself. I read all of the help files on the different types of calibrations, and tried three different methods on my latest set of data. These methods were linear, weighted linear, and non-linear. I found that weighted linear and non-linear were more accurate than linear, but neither provided the accuracy that we really need. Today I’m going to try another type of fit that forces the calibration line through 0. I am also going to try plotting a graph of concentration versus intensity on my own, to see how the calibration process works. I’m also going to make another calibration series that is equally spread over the range we will be working in, instead of the logarithmic set we have been using.

Today I began by combining 3 nucleosome samples together and using the spectrometer to see how good the sample was. Unfortunately it wasn’t a good sample and I kept getting this weird peak in my data at 300nm and then had a slow decline to 220nm. So we skipped those nucleosomes and I tested two more samples that we had. The first had the same curve and the second had the curve we wanted but it was already diluted too much to be of any use to us. After that i tested the last two nucleosome samples that were in the freezer and the first one had kind of the curve we wanted but it peak was from 280-240 nm. The last sample had exactly the peak we wanted but the professor had no idea what was in it. He decided we would go over to the biology department and test the DNA to see how many base pairs it had but we found out we were missing a chemical we needed to do the test so we have to wait. For the rest of the day i spent time gathering supplies and reading up on a similar experiment to the one I’m doing.

Today I took a break from experimenting after yesterdays hectic day. I instead did some reading especially on the diffusion technique I am going to use tomorrow with the old nucleosome samples that we have in the freezer. I am going to use the Mg samples that I made a few weeks ago and run them through the nucleosomes in a centrifuge and measure the amount that is absorbed by the nucleosomes.

Today I finished preparing my new calibration standards, as well as preparing another sample set. I am running the calibration standards for both the calibration and as a sample, so this should help me investigate how the calibration process is working. The new sample set should help to see if the calibration is helping to reduce the error. I am also trying to use a nonlinear fit for the calibration, to see if that helps. I am in the middle of analyzing the data now, but it is unclear whether I have been successful yet or not.

Yesterday we went to a pretty crazy farm to get our chicken blood. The experience was crazy what with the way they killed the chickens, the machine they used to get the feathers off, and the chanting women in a factory line plucking the rest of the feathers. After we got back to campus we began by separating the blood evenly to seven tubes and then putting them through a centrifuge at 2000 times g. after ten minutes we took the samples and removed the plasma, the yellow liquid at the top that had been separated from the blood. We then carefully removed the white coat that sat on top of the red blood cells. This was a lengthy process because it involved us washing the blood with a solution the professor made, running in the centrifuge, and then carefully removing the wash and whatever white stuff we could. After doing this three times we were finally down to just red blood cells and we put those into new tubes and froze them at -70 c.

Yesterday, I spent the morning analyzing the results from the previous day’s test. The results were somewhat inconclusive, but they did give me one useful piece of information. In general, when I used the 1000uL pipette twice, the results were slightly more accurate, by around a percent. The results were still off by approximately 3-4% though, and even the samples that I set to match the calibrations standards were off by this percentage. I tried altering the calibration method, which lowered the uncertainty a bit, by around 1-2%, so I believe the error is coming from both sources. In the afternoon, I designed and began preparing a new set of calibration standards, which go over a larger range and with more points in between. This should help me to minimize the uncertainties.

At 10:00am this morning, Travis and I arrived at the Berry Blossom Farm. We went into their unassuming store front, the entire farm complex smelling of chicken (and I don’t mean the fried kind). In the store, I asked for Alden and told them that we were hear to collect chicken blood. She seemed to have been informed of this possibility and went back to get Alden.

Alden came out dressed in quite messy clothes and a rubber apron. He said that we should come on in and get our blood. I informed him that our equipment (basically a styrofoam container filled with ice, 2 beakers, 50ml of 6% sodium citrate, and cheesecloth) was back in the car. As I walked back, I took a cue from Alden’s appearance and removed my outer shirt so I was just wearing my undershirt. This was to be one of the smarter things I did today.

Alden escorted us through the maze-like complex until we got to a small room. The first thing I noticed about the room was the intense smell. It had an intense animal smell (imagine a room with 50 wet dogs) combined with undertones of blood. The scene seemed to be complete chaos with birds in various states of life and death (alive, dying, dead, plucked). A man who was doing the slaughtering moved quickly removing the dead chickens, putting in new ones, and rapidly dispatching these new be-coned birds with a swift twist of the knife in their throats.

A steel fixture with four steel cones each containing a dying/dead bird. Under the cones was a trough that caught the blood. To the left was the plucker. Basically, this consists of a washer machine type contraption, only there are numerous plastic “fingers” that stick out and remove the feathers as the chicken tumbles around inside. Due to the presence of this machine, feathers were continuously flying throughout the room.

Once we got inside this room, the chaos was infectious. Alden used a garden hose to wash down the cone we were going to use. I quickly asked Travis to pass me the largest beaker and the sodium citrate (which prevents clotting). I noticed a naked chicken come flying out of the plucker and land on the floor, I don’t know if this is by design or if it simply escaped the plucker’s grasp. I dumped the sodium citrate in the beaker. The next chicken was loaded; I put the beaker underneath the chicken and with a quick stab and a twist of the butcher’s knife, the blood started flowing. I collected the slow drizzle of chicken blood. It took three chickens and about two minutes to collect 200ml of chicken blood, even with the butcher turning the head of the chicken so I could get every last drop. One minute through the collection, the women in the next room in charge of the last bit of cleaning of the chickens started singing. (Travis thought it sounded like chanting.) The clear tones of the women’s voices clashed strongly with sight of chicken heads and blood on the floor.

Travis and I exited the building. We poured the chicken blood through a cheesecloth to remove the feathers and other various non-blood elements and put the cleaned blood on ice. As we were leaving to go back to Gettysburg to extract the red blood cells from the whole blood, we were stopped by Alden.

“Do you have a way to keep a chicken until you get back?” he asked. “We have a few extra.”

“Sure,” I said. So Travis and I returned to Gettysburg having bringing back some chicken blood, a frozen chicken, and experience we probably won’t forget for a while.

This morning I finished the RCR training modules, and we also had a training session on chemical safety. During the afternoon, I prepared and tested my two new magnesium series. Each series has 9 samples, ranging from 0mM Mg to 4.11 mM Mg. One series was diluted using the 1000uL pipette, and the other was diluted using the 5000uL pipette. Also, several of the data points should match up with the calibration standards that we use. This should help me identify if the uncertainty is coming from the solution preparation or from the machine’s calibration. Tomorrow I will be able to analyze this data and possibly design a new test if necessary.

Today was another relaxed day I studied up some more on the nucleosome and the histome octamer which is what the DNA wraps around. I learned that as well as the hockey puck shape that the histome octamer can have there is also a sort of V shape with two spheres at the end. I also tried to set up a group file between the three of us so we could post our work and be able to access it anywhere but the gettysburg system got the better of me and we will have to have IT set that up for us too. Tomorrow we are going to go get the chicken blood and then create our nucleosome samples.

Yesterday in the morning, I did a bit more research into how the plasma torch works, as well as the rest of the spectrometer. I gave my presentation on the work that we will be doing, and I also listened to what everyone else is up to. Then, in the afternoon, I worked on preparing a new set of samples to test in the spectrometer. This set should help me understand where some of the uncertainty is coming from. I am going to run two more series, which include some points from the calibrations. This should give us some idea if the calibration method is affecting the results. I am also going to prepare the samples using two different pipette methods, which will test another variable. Hopefully this will give us some idea where the uncertainty is coming from.