Category: Uncategorized

Today, we took a step ahead: getting the NCP to run through the gel almost perfectly. Looking at the picture beside, the first column is the DNA ladder and the bold band at the middle stands for 100 base pairs. But we need about 150 base pairs so we can count the bands after the 100bp band to see the 150bp mark because the bands in increments of 10. The next column stand for the NCP digest for 0 minutes, the next for 5minutes, then 10,20 up till 50 minutes. If you notice closely, there is a thick band on the same level of the 150bp on the 5minute digest column. This is an indication that 5 minutes was the adequate time to digest the NCP.We do not use the other columns because there is a lot of smearing of the whitish band and this indicates over digesting. Therefore we digested our NCP for only 5 minutes, and with the packing ready, we are currently running our NCP through it to separate the mono-nucleosomes from the di-nucleosomes using the size-exclusion chromatograhy technique. This way, the mono-nucleosomes we actually need run faster than the di-nucleosomes through the packing and we can collect them. The NCP has not finished running through but I will keep an eye on it till it does.

Today I looked at the same 5mM Co series, but at a much higher concentration. Using the results I got from the dilute run, I designed a series that should encompass all the concentrations I will be seeing. After running at a higher concentration, the results look much more consistent. Actually, with the exception of the fourth sample, they look very clean and there is clear trend. It resembles the trends we were seeing last summer, but with the Mg taking longer to overcompensate for the Co. Tomorrow I will run three more trials to look at this series closely and get some averages.

Today I spent the day looking at the 5mM Co series, which goes from 0 to 50mM Mg. I was somewhat unsure as to the concentrations of ions that I would find, so I made a very broad calibration series, and a 100X dilution of the samples to test this out. The results were pretty similar to the series I have run before, but with much more DNA. This makes sense, because our collaborator changed his procedure slightly and prepared the samples with more DNA. It is strange that we did not see an increase in the other ions as well, but it may have something to do with the initial conditions we are looking at for this series. Overall, this dilute run was somewhat unreliable, with particularly strange results for the fourth sample. Tomorrow I will use what I have learned about the concentrations to make a concentrated run.

Today, I was handed two new articles to study. These articles had to do with the extraction of the red blood cells and then the NCP form the chicken blood.
The first one was named ” Isolation of histones and nucleosome cores from mammalian cells”. It summarized the extraction of the NCP from mammals but it was related to what we are doing with the chicken blood so I learned a few extra things.
The other article named ” Salt induced transitions of chromatin core particles studied by tyrosine fluorescence anistropy” was actually where professor Andresen extracted our protocol for extracting the NCP from the chicken blood from. So far,I have learned the roles of the different buffer solutions and also the principles behind all the procedure. I will continue with this article and hope I finish reading it tomorrow .

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.