Today I spent most analyzing all three series of data from the three PEG series. Our collaborator, Xiangyun Qiu, was visiting for the day. In addition to providing insight into the results we have obtained, he also provide two more series without PEG to analyze. These series have 5mM Co with Mg from 0-50mM, and 10mM Co with Mg from 0-100. These series should nicely round our results from last summer, which looked at lower Co concentrations.
Today was more of a relaxed day as we made sure everything we set up was running well. First, we made sure the packing was running well and then Professor Andresen set up the nucleosomes to run in the gel. It should be done by tomorrow and hopefully the packing is done too so we could start running some tests.
Today was a mellow day for the Kiwi. The foreign factor were split for the first time today, Fash headed to the farm to harvest chicken blood and the used the centrifuge to isolate the red blood cells. I was left to tend the column that has been 3 days in the making now. The buffer solution is moving through the column so slowly that the lowest setting on the feeder pump still results in a 1cm per hour increase in the column. The day was made faster by reading papers on chromatin structure.
Today we we met up with Professor Andresen’s collaborator , Xiangyun Qiu, from George washington university , to go get some fresh chicken blood. We got the blood from a farm about 20 minutes away from a farmer named Beau Ramsburg.(I heard he makes good sausages.)Here is a link to his website.
http://www.rettlandfarm.com/.
We then got back to separate the red blood cells from the rest of the blood. We went through a long process centrifuging and extraction to finally get about 64mg of red blood cells from 400ml of chicken blood.
On the side, we made sure the packing we were preparing was running well and did not dry up.
Today I ran all three concentrated series that I prepared yesterday. They yielded some interesting results. As the concentration of PEG increases, the amount of DNA precipitate remains somewhat constant, the Mg concentration decreases, and the amount of Co increases. This leads to high overcharging at low PEG concentrations, and around a total charge of 1 for the rest of the PEG concentrations. Looking further into this data should give us more information about the ions behavior.
This morning, we gave our paper presentations. For the rest of the day, my main task was to prepare the remaining three trials for the latest series. I accidentally left calibration solutions out overnight, so I also had to remake those as well. Tomorrow I should be ready to run all of the three remaining series.
Today I prepared and ran the first trial of the new series, which was precipitated in 2mM Co and 20mM Mg. Using the approximate concentrations I got from the dilute trial, I also made a new calibration standard. I will be able to analyze the results more tomorrow. I also spent some time today preparing for a paper presentation tomorrow, which I will be making on how osmotic pressure depends on DNA and ion concentrations in solution. They found that at low DNA concentrations, the ions in solution do affect osmotic pressure, but at high DNA concentrations, the concentration of DNA contributes more to the osmotic pressure.
I won’t beat around the bush – Today was a lesson on why undergrads should not compound their mistakes. It began well with all processes inline to finalize the extraction of nucleosomes. However, I allowed the beaded solution to run too long, and it dried out. So at the beginning of the 3 hour processes to restore the solution, the delicate tube was cracked, setting us back even further. The only consolation was that the separation of the DNA experienced a set back, so there will be a chance to catch up tomorrow. Until then, time to read. Foreign factor out.
Today , things did not go as planned. First we discovered that the separation beads we left in the cold room had dried out. We had left the beads in the cold room with the intention of getting it ready for the NCP that was currently running through the gel. So we decided to re-run the beads but in doing that, we broke the tube in which the beads were in. As we quickly tried to retrieve what we could of the beading solution, Professor Andresen told us that the gel did not run properly and we would have to repeat it. This means that we have to wait till tomorrow to proceed with the NCP procedure. Tomorrow will be a better day!
Today, we continued with the extraction of the NCP from the chicken blood. We were able to get about 15mg in total and now the solution is sitting in an apparatus that uses gel to separate the DNA into base pairs with differences of 10.
