W7D4

Today we looked at the latest results of the NCP experiment. These came out much better, and the concentrations were extremely consistent. They showed some interesting trends, mainly that there was a range where the Mg compensated for all the charge of the nucleosomes, but did not cause them to precipitate. Our main problem now is the large amount of uncertainty we are seeing. We will try to drive this uncertainty down with future tests.

W7D3

Today we analyzed the data from the NCP experiment. The results are still very inconsistent, and are not showing any clear trends. They are showing some inconsistencies in all of the concentration values. Because of this, we prepared for another test this evening, which we can look at tomorrow.

Week 7- Day 3

The results we got from running the two buffers where a little inconclusive due to some issues with the magnesium concentrations in the buffer I made and some other complications with Ben's buffer.But,we could still see the precipitation pattern of the NCP as the MG concentration increased. So Professor Andresen decided we needed to repeat the experiment to find out what as going wrong with it. However, this time, we spun the NCP eight times with the buffer because Professor Andresen suspected that the four spins we did for the last three experiments were not enough. We will analyze the results tomorrow and hopefully we get conclusive results this time.

W7D2

Today we had a pretty big day. We ran two sets of NCP experiments, which involved running the machine well into the night. We are having some problems with inconsistent Mg concentrations, which we hope to solve with future trials. We will be analyzing the data tomorrow, as well as preparing future trials.

W7D1

Today I ran two trials of the buffer solutions for the "5mM" Cohex series. The results leave me a little skeptic that the DNA was actually precipitated in 5mM, because all of the buffer solutions contained pretty exactly 3mM. This would explain why the Mg values only range from 0 to 50, which was the same range used for the 2mM series last summer. I will look into the other buffer solution later this week, to see if it contains 5mM or 10mM.

Week 7- Day 2

Today, we did got some good news and started working towards better news. Professor Andresen got the results of the gel he ran yesterday and found out that we had a good amount of NCP in our sample. Next, we found out that the test we ran with the spectrometer to monitor the ions present in our solutions was somewhat positive. There were a little off-concentrations in the solutions I made but ultimately, we noticed that the amount of phosphorus in the solution dropped steadily as the concentration of Mg in the solution increased. This is good news because it means that the NCP's are falling out of solution(condensing) as they contain phosphorus. To get better results, we decided to run the same tests again but this time with two different buffer solutions. The same one I made yesterday and a new one containing Tris and Nacl without Mg. We will analyze the results tomorrow.

Week 7 - Day 1

Today we got the NCPs ready to run in the spectrometer. Last week friday, we had a series of buffers with different concentrations of Mg in it for the eight different samples of NCP we were going to work on.First we spun down the NCP we had in a centrifuge three times in a buffer that contained MgCl. We then collected the NCP from the filter and tested the pH of the NCP to see if the buffer was effective and indeed it was because the concentration remained at 7.0. We diluted our eight magnesium samples, NCP, and and the buffer we used in the third spin by a dilution factor of 60. Then put them in the spectrometer so we could test the amount of Mg ions in each. We will analyze the results tomorrow. Here is a picture of the test paper we used. The yellowish colour represents neutral and as you can see , the first 8 dots represent our 8 samples and the bigger yellow dot represents de-ionized water which is neutral. Since they have the same colour,we can conclude that the buffers where neutral.

W6D5

Today I took a somewhat short day, with a planetarium show in the afternoon. In the morning, I tried to catch up on some reading. I read one article on the competition of monovalent anions, which was helpful because it tells us we can safely substitute out Chlorine for something that is more measurable. I also got ready to start running the buffer solutions, which will give us an exact look at the solutions in which our DNA was precipitated.

W6D4

Today I ran the remaining three trials of the 5mM series. These results confirmed that there is something wrong with the second and fourth samples, but other than that they were very consistent. The results were also strangely linear, which we saw some of in our results last summer.

Week 6- Day 5

Today, finally finished separating the di-nucleosomes from the mono-nucleosomes as I explained yesterday . Looking at the picture on the right,you can see some peaks on the graph . We are not sure yet which peaks represent what nucleosomes at this point but we suspect that the first pick to your right is where the mono- nucleosomes fall because a test using the UV based spectrometer showed a high concentration of NCP. We will run the different samples at each peak through the gel on Monday and then compare them again to the DNA ladder , so we can finally say for sure what we have.

Week 6- Day 4

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.

W6D3

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.

W6D2

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.

Week 6- Day 3

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 .

W6D1

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.

Week 6- Day 2

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.

Week 6 Day 1

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.

Week 6 - Day 1

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.

W5D5

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.

W5D4

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.

W5D3

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.

Week 5 day 4

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.

Week 5- Day 4

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!

Week 5- Day 3

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.

Week 5 day 3

Today we continued the NCP prep. We found we had recruited around 15mg of NCP particles from the 3ml of chicken blood. Part of the NCP solution was then processed using different techniques to eliminate the protein core and is currently sitting in a gel apparatus that separates DNA chains by lengths of 10bp increments. Tomorrow the process should be completed.

Week 5 - Day 2

Today, we kicked off preparing a new sample of NCP right from scratch using the red blood cells from the chicken blood gotten from last year as the source. We decided to do this because of the abnormal test results we got from using the NCP solution from last year. A suggested explanation for the weird behavior in which the NCP did not aggregate was that the sample may have been mishandled which led to its damage. So we started with the long process of separating the NCP form the cells. We started of with the erythrocyte lysing, then the nuclei lysing and finally the nuclei digestion. We ran out of time mid way through the process but we will hopefully finish up tomorrow. Pictures of solution at different points of the extraction are shown above.

W5D2

Today I prepared and ran the other three trials of this second Tris and Mg series. When looking at the averaged results, there are several important things to note. First of all, there are several strange outlying points. In one sample, there was an abnormally large amount of magnesium. In two others, a strangely large amount of DNA precipitated, but there was little change in the ion concentrations. I will be looking more into what caused these random points. In general though, the results are very consistent and show an interesting pattern. As the PEG concentration is increased, the amount of DNA slowly decreases, and then towards the end it rapidly decreases. The Co and Mg concentrations stay fairly consistent over the range, which leads to a huge spike in contributed charges at the higher PEG concentrations. For the most part, the contributed charge by both ions adds up to around one, but when the spike occurs, the total goes up to above two. Some of this strange behavior could be coming from the comparatively large concentration of Tris?

W5D1

Today I prepared and ran a trial of the next series, which contains magnesium and Tris as well as cobalt. This also involved making another calibration series, with a slightly smaller range than the last one. These different ions in the solution should lead to some interesting effects.

W4D5

Today I analyzed the four concentrated trials I ran yesterday. Overall, there were a few interesting points that I found. Within the range of our error, there was no magnesium in any of the samples, which makes sense because none was added to begin with. There was a fairly consistent amount of Phosphorus (DNA) in all of the samples, which ranged between 500 and 600 ppb. In general, as the concentration of PEG was increased, more Cobalt was precipitated. Logically, this led to an increase in the charge contributed by the cobalt at higher PEG concentrations. At the highest PEG concentration, the charge contributed was around 1, which raises the question what was compensating for the charge at lower PEG concentrations.

week 5 day 2

Today we departed from the frustration of testing the aggregation abilities of old nucleosome samples and started from scratch. We are currently practicing the technique of NCP prep, or in other words extracting the nucleosomes from chicken blood. The process takes over a day, today being spent using a tris, NaCl, EDTA and PMSF solution and centrifugal techniques to reduce the chromosomes. However, an overnight process if chromatin dialysis is essential. So we will pick un tomorrow with chromatin.

week 5 day 1

Today resembled Friday. A re-run of Bertin's NCP aggregation was attempted, however, results did not resemble Bertin's. Large concentrations of Magnesium were approached in the NCP solution, however, no significant NCP was observed to be aggregating. One suggested problem was the sample was old and poorly handled, resulting in the essential tails of the NCP being degraded. Next week a better sample will be used.

Week 5- Day 1

Today, we repeated the same experiment we did last time in which we added MgCl to the solution of NCP so we can observe the reduction in concentration but we did not observe that. We decided to then make a new solution of NCP from chicken blood starting tomorrow and then we will repeat the experiment so we can spot out where we went wrong with it.

Week 4 Day 5

With yesterdays data in disagreement with Bertin's experiment, today was used to determine the reasons for these discrepancies. We began by adding surplus amounts of Magnesium to the solution in an attempt to see some form of NCP falling out of solution. This was to no avail as the solutions continually tested a similar concentration of NCP from UV vis spectrometer testing. The reasons why this experiment is not consistent remains unknown. Perhaps the Magnesium was incorrectly concentrated. The experiment may need to be performed again.

Week 4- Day 5

We added more magnesium to the NCP solution to see if aggregation occurs but after testing the concentration of the NCP after doing that, we found out that the concentration remained the same.. However this is not meant to be so. The addition of the magnesium salt should decrease the concentration of NCP in solution. We will look into this on Monday

Week 4 Day 4

Today was spent attempting to simulate Bertin's NCP aggregation experiments. Magnesium was repeatedly added to a 1mg/mL NCP solution at the increments indicated in the previous blog. Unusually, no significant change was observed in the NCP concentration as a result of Mg2+ addition to the level of 15mM. Futher investigation into the reasons behind this disagreement with Bertin's experiment will be perused tomorrow. The experiment may have to be repeated.

Week 4- Day 4

Today, we went ahead to perform the experiments with the solutions we prepared yesterday. We added amounts of magnesium to the NCP solution and then tested the concentration of the NCP in solution after each addition with the UV based spectrometer. The expected result was for the concentration of the NCP in solution to decrease because the magnesium should make some of the NCP aggregrate and fall out of solution. However this was not the case.So we decided to add higher concentrations of magnesium ton the NCP solution tomorrow and see what happens.

W4D4

Today I looked over the results from yesterdays test. This time, I found that none of the samples contained magnesium, which is different from what the dilute series showed. Everything about this first test seemed very solid, so I made three more concentrated series to test. Together, these four series should provide a very accurate look as to how the ions behave in these samples. I will be able to compare them more tomorrow.

W4D3

Today I spent a very large amount of time making a very accurate calibration series. This involved double checking all of my solutions with an accurate scale to make sure the pipettes were being sufficiently accurate. Using this, I was able to able to measure very accurate amounts of the stock solutions, and make a much better series. In the afternoon, I made a concentrated set of DNA samples and ran them with my new series.

Week 4 - Day 3

Today, we prepared solutions in order to try to replicate Bertins experiment in which increasing concentrations of Mg are added to the nucleosome solution to see how the nucleosome aggregraates at the different concentrations.First we made a 10ml of a 1M solution of Magnesium chloride. Then we prepared the nucleosome solution by adding 67.2μL of the concentrated nucleosome to 10μL of 1M tris and then diluted the solution to 100μL with DI water. It is this solution we will add the magnesium chloride to while testing the concentration of NCP left in the solution after each addition using the UV based spectrometer shown above

Week 4 Day 3

Today was spent creating a protocol for the Bertin experiment and setting up the experiment, so come 9am the work can be pushed through. The experiment will consist of a 1mg/mL solution of NCP particles being immersed in an increasing concentration of Magnesium solution. The 12 different concentrations that will be measured and compared to the concentration of NCP particles will be approximately .2mM, .4, .6, .8, 1.3, 1.9, 2.6, 3.7, 4.9, 6.1, and 8.6.

The purpose of the experiment will be to compare the results to Bertin's results.

Week 4- Day 2

Today, we went to check on the nucleosome solutions we left to filter in the cold room and found out that they were in good condition. We then sorted out the collected samples by figuring out which test tubes had the single and double nucleosomes in them. We then tested the samples containing the single nucleosomes to find out the concentration. We did this using a UV based spectrometer which I will show in my next post. After this, we concentrated the sample by using a centrifuge running at 5000g to collect only the nucleosomes from the solution. We tested the concentration of nucleosomes in the concentrated sample and found out that we lost roughly about 7% of the nucleosomes in the solution probably due to the filter in the centrifuge. But over all it was a good result.

W4D2

Today I spent the entire day looking at the results that I measured yesterday. There was a lot of information there, and it took some time to modify my spreadsheets from last summer. Two of the series displayed clear patterns, and one of them seems somewhat random. The series with a large concentration of Tris has a clear spike at higher concentrations of Tris. The series with a large concentration of Mg has a clear spike of Mg at lower concentrations of Tris. These patterns should be interesting to investigate with future trials. Tomorrow I will be making what will hopefully be a very accurate calibration series to run some concentrated trials.

W4D1

Today I spent the morning reading several articles on osmotic pressure. I am trying to get a better idea of the results I should expect from the new PEG series I am running. In these tests, the main variable that we will be testing is osmotic pressure. The best idea that I can build as of now is that osmotic pressure has to do with the concentrations of solutes in a solvent. It has several different definitions, depending on the context in which it is being used. The one that seems most relevant to our work is the energy density of solvent molecules. The idea that I am getting is that PEG takes up space in solution that used to be available to water.
I spent the afternoon running my three dilute series after the nitrogen arrived. I will be able to analyze these tomorrow.

Week 4 Day 2

Today we revisited last years NCP samples, from Xiangyun Qiu. The day began with a nervous visit to the cold room where we found a good set of UV data from a sample of NCP solution that was left to separate the night before. The picture to the right shows this sample, where particles in a solution were separated through a beaded solution by size. The high spike corresponds to the solution that contained the NCP. Using this graph we were able to pull about 50ml of NCP from the sample, a tone for the experiments we will be running in the future.

From here we centrifuged the NCP solution to create a highly concentrated solution of NCP. Tomorrow we will be using the sample to recreate Aurelie Bertin's experiments of observing the changing concentration of NCPs in varrying cation (Mg) solutions.

Week 4- Day 1

Today, we moved the UV monitor and the fraction collector down to the cold room in the science center so we could filter the nucleosome samples from last year using the chromatography I talked about early last week. We had to monitor the water level in the test tube and make sure it did not dry up so we could then add the nucleosome sample into the tube to start the process. Here are pictures of the apparatus

Chromatoraphy stand with beads in it.

Dilute stock solution

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Fraction collector showing improvised cord

W3D4

This morning, we had a meeting to discuss both our goals for the next week, and a series of papers that we read. I presented the paper on osmotic pressure and viruses. For the rest of the day, I prepared myself to run the three new series next week when the gas tanks get here. I prepared dilute samples of all three series and a calibration series to go with them. On Monday, assuming the gas gets here on time, I will be able to get preliminary results for all of the new series.

Week 3-Day 4

Today, we were involved in various activities in the lab. First we kicked of the day with presentations on pre-chosen articles. I talked about the electrostatics that has to do with the nucleosomes in general from the article " DNA inspired electrostatics".

Next, we continued trying to figure out how the optimum level at which the water we feed into the "chromatography" test tube comes out at the other end at the same rate in order to prevent the "beads" from drying up.

Next, we prepared the stock solutions of the NaCl and Triz solutions. Professsor andresen showed us,using the NaCl, how to go about measuring the right mass of the solutes and also the correct way to dilute them with the solvent( water) and then finally how to filter any dirt from the solution. Ben and I then proceeded to do thesame for the Triz solute .

Lastly, we had to dilute the stock solutions we made to a 1 litre solution. We did our calcutations and verified with Professor Andresen before proceeding with the dilution. We are looking forward to next week when we get deeper in the research!!

W3D3

Today we spent the morning preparing a talk to give the department describing the basic theory and motivation behind our work. We described how our work applies to the protein-DNA interactions that occur naturally in the body many times per second. Our work also can be used to package DNA in useful ways and facilitate its uptake into cells for gene therapy. I also spent a good deal of time reading over the paper I will present tomorrow on osmotic pressure's effect on virus DNA ejection. The virus system does not exactly apply to our work, but a lot of the concepts, including osmotic pressure and DNA-DNA interactions are very similar.

W3D2

Today I spent most of the day reading through the first half of Prof. Andresen's PhD thesis. His work uses anomalous small-angle x-ray scattering (ASAXS) to describe competition between different valence ions in the ion atmosphere surrounding DNA. I found his background of the basic biological theory behind the work especially useful, because it was laid out in a very clear manner. He also described the motivation behind the work we are doing, which was also very interesting.

Week 3- Day 3

Today, We had a presentation where Ben,John and I explained what our research was about to all other students involved in research with other professors. We talked about the motivation of our respective researches.

I also had to prepare for our article presentation tommorow. The Andresen team all have to pick an article each and explain to the other members of the team. I picked the article " DNA inspired electrostatics"

Lastly, we finished working on the "packing" of the beads I talked about yesterday by making sure they did not dry up in the tube. I will post the picture of the apparatus used for better understanding .

Week 3- Day 2

Today, we had to improvise for a cord needed to run the UV monitor and the fraction collector. So we got our hands dirty with connecting wires and I also got to solder for the first time! The connection using the improvised cord worked as planned with some help form Prof Andresen. We also used a form of chromatograhpy to separate some mini beads from solution. We will use these beads later on to separate the single, double..etc chromosomes from one another and then analyze them using the fraction collector and the UV monitor.We also got familiar with the new labbelling device which we will use to label our test tubes from now on.