Week 10 - Day 3

Today, Ben and I finished working on our slides for our presentation to cap off the research. Based on suggestions from Prof Andresen and John, we made changes to our data arrangement and delivery. We did a practice session with John spotting out more ways to improve the presentation. We were finally ready but the end of the day and gave our presentation. I think it went pretty well and we are now assured that the rest of the Physics research have a good idea of what we did during the summer!

Week 10 - Day 2

We got the results today and it was another disappointment. The Na concentration was fluctuating while the Mg concentration seemed better but not still up to expectation. Professor Andresen decided to check the parts of the spectrometer again to see if there is any problem wrong with it and he also personally made some solutions to see what happens. Ben and I also started working on our slide show for the presentation we have tomorrow. We are gathering our data and doing some final analysis on them.

Week 10 - Day 1

Today, I made buffer solutions once again and tested them in the spectrometer. The results will be analyzed tomorrow as we are going to run it overnight.

Week 9 - Day 5

I remade the solutions using the right concentrations of Mg today. The results we great! What a sense of relief. All the prepared concentrations fell with 1% of their intended concentration. So we decided to step it up one notch. Professor Andresen suggested that I made more solutions in ppb But this time I should make two sets: One with only Mg diluted with water and then the other one with the complete components ( tris and Nacl). I made the solutions and stored them in the fridge so we could test them on Monday and analyze the results.

Week 9- Day 4

Today, we ran and analyzed the results of the solutions I made in ppb (parts per billion). There was another problem today as the results showed that there was no Mg in the solutions at all. I checked through my calculations and found out that my calculations were off by a decimal place which made my calculations for the Mg concentrations 1/10th of the actual values they should have been thereby making them undetectable by the spectrometer. I decided to repeat making the solutions tomorrow.

Week 9- Day 3

Today, we got the results for the solutions I made yesterday. We had both good and bad news. Good news first: The Na concentrations we almost on points as we had only two samples falling of the 10mM concentration it was supposed to be(7.9 & 7.7mM).The rest were about within 5% of the 10mM intended concentration. However, the bad news is that the Mg concentrations were all off by a factor of 7. This was surprising and confusing at the same time because I was pretty sure I did not multiply my calculations by any factor at all. So, the conclusion today was that I should make some solutions of just different Mg concentrations and water without the tris and NaCl. I made about 5 samples starting form an Mg concentration of 300ppb with an increase of 600ppb between solutions.

Week 9 - Day 2

Today, I took special care in making the solutions. I weighed them instead of the normal approach of just pippetting the needed volumes into test tubes. I also talked to Professor Andresen about possible problems with the whole experiment and we decided that we wait for the results of this run and then we can change our approach to making the buffer solutions.

Week 9- Day 1

After the little success we got, I decided to make more solutions today. However, the results are not looking good. The next step will have to be for me to weigh them instead of just relying on the pipette to measure the amount of solution that I put into the test tubes.

W9D1

Today I ran two large sets of samples, one for myself and one for Fash. Fash's samples were his latest set of buffers, for which we have high hopes. After fixing our problems last week, these results should be very consistent. We will look at the concentration data tomorrow. I finished running all of my Cohex 10mM series, and the preliminary results show a clear pattern. As expected, at high Mg concentrations, very little DNA precipitated, so the results are somewhat unreliable. Tomorrow I will double check the buffer solution, and begin to write up some summaries of our data.

Week 8

Last week was a busy week, which kept me from posting much. I'll try to give a somewhat decent account of what happened, and the status of our projects. Last week, we had some trouble getting consistent results from Fash's buffers. We investigated a variety of variables, from my calibration technique to pippetting errors. We also had a problem with counts from the spectrometer. We eventually discovered that part of our problem may have been coming from the plasma torch area being dirty. There was also a possible problem with consistent nitrogen flow. We now think we have most of the issues figured out, and should be able to proceed with collecting consistent results. Ben has been getting consistent buffer values, and is proceeding with his NCP experiments. For my project, last week I ran the second buffer solution, and began to test the actual samples. The second buffer solution had somewhat surprising concentrations, because they came out to consistently around 8 mM Cohex, when we thought they were 10. I am going to double check these results after I test the actual samples further.

Week 8- Day 5

I prepared more solutions today. But since John was leaving early today I kept them in the fridge so we could run them on Monday. Hopefully they turn out good!

Week 8 - Day 4

Today we tested johns results, and although they looked better than mine, they are not as close as we wanted them to be. So we were left wondering what was wrong with either the machine, the stocks or us! Well, we ruled the machine out because Ben's results were good. We were on the verge of ordering new stock solutions when professor Andresen decided to check the sprayer in the machine( spectrometer) and found out it was kind of clogged and worn out. He changed it with a new one and we hoped that as the problem with the machine. So I made some more solutions are we quickly tested them. Surprisingly we got really good results but there was a little problem because the measured concentrations of Mg we measured were in multiples of 10 of what I actually prepared. This might have been a mistake on my part so we decided to make more solutions tomorrow.

Week 8- Day 3

We tested and analyzed the samples I made yesterday. They still turned out random. So John and I decided that he made a few samples of his own so we could conclude if the problem was with my measurement or the stock solutions themselves. John made solutions with magnesium concentrations of 1mM and 2mM. We will test the results tomorrow.

Week 8 - Day 2

After the conference we each have some new assignments. Mine - to test the ionic attachment of Magnesium to NCP. It's simular to the last tasks, but a different method. Instead of adding Mg solutions of different concentrations to NCP, we are adding NCP to pre tested concentrations of Mg solutions, and testing the difference between the two. The goal is to get really careful and accurate data, so yesterday and today was spent working on the method. Yesterdays concentrations had a 6 sample standard divination of 1.4%, hopefully today's samples will be even less.

Week 8- Day 2

We analyzed yesterdays result in detail and decided I needed to make some new buffer solutions. I took more time and did it more carefully so the results should be a lot better than yesterday. We will analyze the results tomorrow.

Week 8- Day 1

After a week long break, we all gathered together today to discuss. Professor Andresen gave us a little briefing of how the conference went and he proceeded to let us know about future experiments we were going to carry out. The first problem we wanted to correct was the ability to make solutions to a good accuracy. So , I made buffer solutions of different Mg concentrations. If the results do not turn out well, I will have to make some more tomorrow too because we are gunning for perfection with this.

Week 7- Day 5

Today was a short day for me with Ben and John away. I helped Professor Andresen sort out some articles for his conference. I also went through some of them as they had some relevance to what we are doing too.

Week 7- Day 4

Today, we got the results of the last test which turned out pretty well but with some unusual uncertainties. However, the got some good results on the concentrations at which the precipitation of the NCP occurs. I also helped Professor Andresen do some spins with some new NCP samples. The only difference with this samples was that the H3 and H4 tails were cut off from them. Professor Andresen was also preparing for his conference next week.

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

.

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.

W3D1

Today I prepared a new calibration series, and some more DNA samples to run. We ran these samples under the supervision of a PerkinElmer tech, and took away some important lessons. We learned that altering the current through the plasma coil can give us higher counts for elements that require ionization. We also learned that slowing the flow of sample through the nebulizer will increase this effect. I prepared a new calibration series to run new PEG samples tomorrow.

Week 3 Day 1

After a memorial weekend of reading up on the theoretical models of the binding of cations to DNA, the lab took a day to tidy and read more on what the future holds. A lab tech came in to assess the OES and answer any questions we had. Hopefully getting our hands dirty in the lab tomorrow.

Week 3- Day 1

We had a technician show us better ways of using the spectrometer today. I also started reading another article named Electrostatic mechanism of chromatin folding which was written by David J. Clark and Takeshi Kimura.The article talked about the behavior of the nucleosomes in a salt solution of NaCl with the cations also present in the solution. It investigated the behavior with low and high concentrations of the salt solution and the DNA. With kurt back, we will soon kick into a higher gear in the research.

W2D5

Today I spent more time looking into papers on osmotic pressure. The first one I read was by Hansen et al, and it described a new way of looking at ions around DNA called the cell model formulation. This model is more accurate for determining osmotic pressure at low salt concentrations. It says that this "cell" acts as a neutralization volume for the counterions, and the electric field vanishes at the cell wall. Within the cell, the ions follow the PB equation. I also read another paper by Evilevitch (EVIL!) about DNA ejection from viruses. This paper suggested that PEG can be used to prevent the ejection of DNA from viruses. The use of smaller osmotic pressure changing molecules such as Glycol does not prevent the ejection of the DNA.

W2D4

Today I ran another competition between Ben and Fash, aiming to hone their pipette and calibrating skills. I also read a paper on DNA counterions. This paper suggested that counterions can contribute to the macroscopic properties of DNA solutions, such as osmotic pressure. It said that at High DNA concentrations, the counterion contribution to osmotic pressure prevails. Also, this paper suggested that there was a certain DNA/SAlt concentration range where osmotic pressure was proportional to the DNA concentrations, and independent of the salts.

Week 2- Day 5

Today, with Ben away in Phili,I went over the articles we read last week to further strengthen my knowledge in the biology of the research.

Week 2- Day 4

Today, Ben and I wanted to become more familiar with pipetting,calibrating and operating the spectrometer in general. So we decided to compete in making solutions of Mg,Co and P of 0.05,2,4,6,and 8ppms.

We then ran random samples which John provided with our respective calibrations to see which ones matched better. Well, to cut the story short, I won!! Although I have to admit that my Mg calibrations were a bit off. We will be working more on our pipetting skills over the next few days.

W2D3

Today we began with some plans for aesthetic work to be done to masters hall. This involves a lot of poster renovation and updating. I then got out some leftover DNA samples from my work last summer for Ben and Fash to analyze. They used diluted versions of their calibration standards from yesterday to calibrate the spectrometer, and then attempted to determine the DNA and ion concentrations. We found that there was actually a few errors in their calibration series, but they were still able to obtain decent results. I also spent some time today educating myself on osmotic pressure, because many of the series I will be soon looking at deal with PEG and osmotic pressure. I found one article that suggested that divalent cations could cause attractive forces between DNA molecules under proper solution conditions. No such attractive forces were found with +1 ions.

W2D2

Today I prepared mystery solutions for Ben and Fash to identify using the spectrometer. These solutions had random amounts of Mg, Co and P that I had predetermined. I gave them some rough guidelines on what range my concentrations were in, which is around what we have when testing actual DNA samples. They made an accurate calibration series, and were able to identify the mystery solutions with very high accuracy. I also spent some time today looking over a lot of my results from last year, getting ready to start running actual DNA samples again.

Week 2- Day 3

Today, we ran solutions similar to the ones we did yesterday. The only was change was that we diluted the 1,2,4,6,8,10(ppm) solutions to 1,20,40,60,80,100(parts per billion(ppb)) respectively and then calibrated the spectrometer with them. Also , the samples we used this time were some of the DNA samples John used last year.

We also learned more about analyzing the results from the spectrometer. We noticed one point( the 80ppb) on the graphs were out of place. We probably added a little too much of Cobalt in the original 8ppm solutions we made yesterday. Otherwise, every other thing seemed pretty okay.

Week 2 - Day 3

Today the veteran put the foreign powers to the test by pulling out an old batch of DNA and left us alone to test the ions present in the solution. The foregin fact passed fairly sucessfully with most of the data corresponding to Johns previous tests. One of the calibrating solutions (80ppb) had a out lying concentration of Cobalt, which threw the calibration and results off a little. But all and all a good day, and feeling more comfortable using the equipment.

Week 2 - Day 2

Today,John "the veteran" made solutions of magnesium, cobalt and phosphorus of unknown concentrations and our task was to use results from the spectrometer to make good estimates of their concentrations. We started off by making calibrations of the same solution varying from 1ppm(parts per million) to 9ppm.

We then ran these calibrations in the machine along sides johns samples so we could use our calibrations to deduct the concentrations of his solutions. This was another opportunity for us to improve our pipetting skills. Ben taught me how to pipette properly after he noticed that I was unknowingly drawing out to much solution each time.

We ended up getting the right answers to the concentrations of johns mysterious solutions.We are looking forward to the next task.

W2D1

Today we began with a lesson on chemical safety. After that, I gave a crash course of the basic chemistry that is required for our lab work. We then each prepared a set of samples, spanning from 1 to 10 ppm of Mg, Co and P. Using my set as the calibration, we tested the other sets for accuracy, and they were both very close, especially for first tries.

W1D5

Today I spent the morning brushing up on some reading, looking at a review article done by Bai, et al. This article described research very similar to my own, because they are also using spectroscopy to look at the ion atmosphere around Nucleic Acids. They did not use precipitated DNA though, rather they use a buffer equilibrium technique to preserve the atmosphere around the DNA. They found that the total ions around the DNA almost always was within error of exactly compensating for the DNAs negative charge. They also studied the competition between different molecules with similar charges, finding that bulkier polyions were often less competitive than the alkali and alkaline earth ions. I then spent the afternoon brushing up on my OES skills, preparing and running a test series and calibrations set.

Week 2 Day 1 - Experiments

As promised, learning the experimental ropes began today. It began with a crash course in stoichiometry, led by John, the student veteran of the Andresen lab. Once we had recalled the number crunching of high school chemistry, we were sent to the pipettes to create solutions of varying concentrations of Mg, P, and Co. We created these samples so we could be trained on the main machine of the lab, the Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). To quickly summaries what this machine does, it pumps solutions through a high temperature plasma, which blasts the molecules in solution apart and detects the resulting ions by analyzing the discrete wave lengths detected at different regions throughout the machine.
Using the ICP, we can send DNA on NCP molecules that have been bathed in different ionic solutions, we can detect the ions that attach themselves to molecules. It's important to note that the main error in the experiments comes from pipetting, so the Foreign Factor will be getting lots of practice on that, competitions galore.

Week 2-Day 1

Kurt was not around today so John was in charge. We had a session on lab safety early on, to start the day. Jeremy Kumar gave us essential information on how to handle situations gone wrong in the lab and he gave us helpful information about appropriate lab behavior.

Next, John showed us how to prepare solutions of Cobalt, Magnesium and Phosphorus. We learned how to use the pipettes and brushed up our old school chemistry skills in stoichiometry as we calculated concentrations of solutions.

The most exciting part of the day was learning how to actually run the Spectrometer as John lead the way!!

Day 4- Last day of reading

We rounded up reading the articles we had to cover today. We read the "Physics of Chromatin" article by Helmut Schiessel. This article was like an all round summary of all the other articles we read during the week. First , it talked about the structure of the Nucleosomes, then proceeded to say that DNA is packed in a way that the information would be easy to access even though it is packed in a complex way.

Next, we read about how an increase in ionic strength makes the DNA thicker and hydrogen bonding being responsible for the linking between the DNA- backbones and the histones surrounded by the DNA.

The paper also confirmed once more that counter ion release was responsible for overcharging. The paper ended by using a sphere ad chain model to explain the DNA and histone interaction in three different cases.
1) weakly charged
2) strongly charged and
3) physiological condition

Day 4 - Last day of reading

With today being the last day of reading and next week the beginning of learning experimentation procedures, it seems appropriate to discus a few of the experiments we have read about during these past few reading days.

With certain aspects of these nucleosome structures fairly ambiguous, such as how they are packed along the genome, investigation into these uncertain aspects of nucleosome function is currently being looked into. The main work we have looked at is the paper, Structure and Phase Diagram of Nucleosome Core Particles Aggregated by Multivalent Cations, by Aurélie Bertin, Stéphanie Mangenot, Madalena Renouard, Dominique Durand, and Françoise Livolant, where they examined the behavior of NCP in various polyvalent ion solutions. The general trend was that when a solution of NCP had polyvalent ions added, the NCP rapidly fell out of solution. However, as more solution was added the NCPs went back into solution until it reached its original concentration, an unusual trend that that could suggest certain processes and structures of chromatin and NCP.

Day 3

Ben has basically said all we did today at the lab and has given a little lecture on the Necleosome core particles(NCP),but i will add a little to that. I will explain a little about the experiment that was done on the NCP he talked about. As he said already, the NCP has a net negative charge and so the experiment involved adding two different cations ,namely a magnesium ion and a spermidine ion to a solution containing the NCP.These ions were added separately in two different experiments. The behavior of the NCP was observed as they precipitated when the ions were added but also came out in re-dissolution when the number of ions added reached a certain threshold. They used different concentrations of the NCP to see the different behaviors it will have. We learned that, the lower the concentration of the NCP was, the greater the interaction it had with the ions. We will keep you posted, but for now the second half of the international factor is out till tomorrow.

Day 3 - Nucleosome Core Particles (NCLs)

Today the foreign factor was churning through papers again, this time looking at prior experimentation done with Nucleosome Core Particles (NCLs). These publications are along the lines of the work us internationals will be doing around the lab, so as we know what its like to be in a strange land, for those of you who are foreign to NCLs, we'll turn this post into a lesson on NCLs and how they act in certain environments.

If you can imagine wrapping a thick string around a cylinder, that is a basic picture of an NCL, with DNA strands being the string and a histone octamer, or protein being the cylinder. As seen in the image to the right, the 'nucleosome' refers to this whole string and ball structure of a 146-147bp (base pair) DNA strand wrapped around the histone. This tiny structure (10nm) has a lesser negative charge than isolated DNA, due to the positive core, with an overall charge of -150. The nucleosomes can be thought of as the building blocks of chromosomes, as multiple nucleosomes can be wound together to form chromatin fibers, which be further arranged to form chromosomes. The reason we are interested in these nucleosomes is that due to the small, dense and complex structures, not much is known about the processes that compacts nucleosomes into chromatin fibers.

We'll let that information be absorbed and talk about experimentation tomorrow. Foreign factor gone.

Day 2 - Studying continues

We moved on to different article that also tries to explain why the liked charged macroions/polyelectrolytes (DNA in particular) will want to stay attached together,in a condensed form ,when counterions are added to a solution in which they are in. We also learned ,from kurt, about the role that enthalpy and entropy play in this procedure. We learned about why the system as a whole chooses to preserve its entropy at the expense of its enthalpy by using its highest mutli-valent ion in the bonding process. I have learned a lot in just two days. It is going to be a great summer!!

first day of "freshmen year" once again!! But for research this time

Today, we basically had to catch up with the readings for research. We have a lot of fun stuff to read and get acquainted with, because the research involves a decent measure of biology. So I decided to get going with it right away and discover why two molecules(DNA) of the same charge will want to sit beside one another holding hands(be attracted to one another). They should be enemies according to the physics we all know. So to get to the bottom of this, Ben and I read our first article and then bombarded Kurt with all our uncertainties(there most always be uncertainties in labs right?)and he made them all clear. Tomorrow,we will be on to the next one.

p.s make sure you laugh at the uncertainty joke. Thanks

Day 1 - Foreigners hit the books

With a summer theme of Bio-Physics, it would seem important for Fash and Benjamin (the Foreign factor of the lab), the young Nigerian and New Zealand aspiring physicist to brush up on the prefix (Bio). Currently, although we are well versed in physical ideas, such as electrostatic repulsion, Fash's and my biological knowledge doesn't extend further then a semester of bio 112; leaving us about as lost as we felt line dancing at the local rodeo last weekend (although Fash did suit his pink rodeo hat).

Today -- and most likely the remainder of the week -- will be dedicated to becoming acclimated with the biological and chemical content of the Andressen lab, and also getting a good grasp on prior work that has been conducted on the unusual properties of DNA.

From the literature we covered today (DNA-Inspired Electrostatics - W. Gilbert, Electrostatics of Strongly Charged Biological Polymers: Ion-Mediated Interactions and Self-Organization in Nucleic Acids and Proteins - G. Wong, L. Pollack) the interesting behavior of DNA came to the forefront of Fash and my bilingual discussions. Switching effortlessly between New Zealandish, English and American, we conversed about the theme of electrostatics. We agreed that one of the most striking aspects of DNA was its charge to length ratio. The long thin DNA coil contians one unit of negative fundamental charge every 0.17nm of length. Even more remarkable than this fact, are the experimental observations that despite the repulsive effects one might expect from such dense, like charge, the DNA attracts itself under a range of solution conditions. This was a recurring theme in the literature studied, with numerous theories including Poisson-Boltzmann Mean-Field Theory, presented in an attempt to explain this unintuitive phenomena.

Also applicable to the work we will be doing was the descriptions of how DNA acts in varying ionic solutions. When placed in "physiological conditions," (1MolL-1 NaCl) the DNA follows the shape of a coil, however, when placed in a highly dilute solution, the DNA forms into a torus (donut) shape with an average radius of 50nm. This resembles the experiments the foreign factor will most likely be looking into. However, instead of dealing with isolated DNA, we will be looking at how DNA, with nucleosomes still present within the structure, will act in varying solutions.

That seems like enough of an introduction, apologies for the accents, and until tomorrow, the Foreign half of team Andressen is out.

The Summer Begins Anew

What an exciting time! This summer we have one returning researcher, John Giannini, and two new researchers, Olayinka O. Fasawe (Fash) (below) and Ben Constable (right). These two will be taking the reins on the nucleosome work that I just got funding for through the Research Corporation Cottrell College Science awards. They will be looking at all of the interesting ways that ions interact with nucleosomes and how these ions sometimes manage to make the nucleosomes behave in peculiar ways. John will be continuing his research on DNA condensation.

In addition to helping them all with this work, I'll be working on a project that uses x-ray scattering to study these same nucleosomes. It should be a busy summer!

But with this talent and the wonderful resources available to us, we're ready for it!