Wednesday, June 30, 2010

June 30th

I finally have real, tangible, reasonable results from our collaborator's samples! I spent the morning making new calibration standards and a new set of test samples. I also started reading an article about questionably-plausible applications of this research in gene therapy. It also has applications in designing safer and more effective drugs, as well as learning how viruses pack DNA so efficiently, which would help us fight them. Anyway, the results look fairly consistent at least, I would say accurate but I'm not sure we know what they're supposed to look like. There may be one outlying point that I need to check into, it's possible that I messed up the preparation. The results seem to be telling me things, I'm just not sure what those things are yet. It is clear from the results that as you go up the series of increasing initial magnesium concentrations, the final results are that the magnesium out-competes the cohex. The unknown part is how and why this happens.

June 29

Today we got back our DNA gel and were surprised to find that each of the trials seemed to be very similar even the zero digestion sample. After a lot of thought the professor determined that the process we did for digesting the nucleosomes earlier was around 100 times off than the recommended so that our samples were not originally digested all the way that we wanted. Tomorrow I am going to run a timed digestion again except this time we are going use more nuclease in order to make up for the digestion that we haven't done yet.

Tuesday, June 29, 2010

DNA Gels and Poor Digestion

As Travis mentioned in an earlier post, today we finished running our gel. With the help of the Steve James lab, we imaged our DNA. The results were not promising.

What you see on the left is just the DNA ladder. Those numbers correspond to the number of base pairs (bp). On the right, in the lane labeled 1, you see our DNA ladder with red lines tracing the corresponding markers. As you can see, we can clearly see the 330bp and 100bp markers. What we can't see are all of those nice little lines in between. This could be a problem in the future, but is small beans compared to the mess further to the right.

In lanes 2-12, we had placed DNA samples that had gone through various times of digestion. What we were trying to do was take DNA that was all kinds of different lengths and snip most of it down to 146bp. Each lane corresponds to 5 minutes later than the one before it. So lane 2 is time zero, lane 3 is 5 minutes of digestion, lane 4 is 10 minutes of digestion, and so on. Now, you don't have to be an expert to realize that lane 2 looks identical to lane 12! This is a bad sign. But what does it mean?

There could be many issues: The gel could have been run incorrectly, the samples could be contaminated, or we could have so much DNA (gels aren't normally this bright) that it somehow makes us unable to see what is actually happening. But I'm pretty sure none of these are the cause.

The procedure for digestion I used was a modification to the procedure I had used the first time I did a nucleosome preparation. The modification required a few more measurements, but it was claimed in the paper I pulled it from that this procedure gave better results (i.e. more nucleosomes at the end). We did these extra measurements (I believe correctly) and added the amount of nuclease (the digestors or eaters of the DNA) that the new procedure called for. However, as both Travis and I found out in later calculations (that perhaps should have been done before the trial digestion) this new procedure called for 100 times less nuclease than the former procedure called for. The final result: No digestion.

Luckily enough a) We were doing a trial digestion, not the real thing, so we didn't lose any real amount of samples and b) It is quite easy to repeat the trial digestion with the old method and then rerun our gel. And that's exactly what we are going to do tomorrow.

Wish us luck.

June 29th

This morning, I analyzed the results from my first tests on the real samples. Unfortunately, the machine was not properly calibrated to work well at such low concentrations. The pattern we were expecting to see was for the magnesium concentrations to increase across the series, and for the cobalt and phosphorus concentrations to decrease. The data slightly resembled these trends, but it was also riddled with confusing negative values. These negative values are what suggest that the machine is not calibrated properly. After lunch, I set out to see if the machine could be calibrated to be accurate at very small concentrations. I diluted my previous calibration series X100, because that is more around the range we are dealing with. This is because we diluted the samples X100 to try and conserve the sample if possible. I ran the tests this afternoon, and from my first look, it appears that the machine works quite well at the single parts per billion range, which is impressive. I should be able to start getting realistic data on the real samples tomorrow. One problem that I did have yesterday was that as I was running the real samples, the plasma torch was burning bright orange, and it seemed much stronger than normal. This is disturbing both because it could be damaging the machine, and it will almost definitely throw the results off slightly. I also got a strange error about mercury lines this morning, but it seems everything is alright.

June 28

Today we ran our DNA samples that had been eaten with the nuclease and then stopped with the EDTA through a DNA gel which measure how many base pairs are present in the DNA. It does the by running a current through the gel and the very negative DNA moves towards the positive end of the current. The smaller the base pair number the faster the DNA moves so the higher base pairs would be at the top of the gel and the lower at the bottom. Because it was running so slow we turned the voltage down and allowed the gel to run overnight, and tomorrow we are going to dye the gel and look at it with a UV light.

Monday, June 28, 2010

June 28th

Today I continued my research into the Poisson-Boltzmann equation, as well as into an experiment done previously by Professor Andresen. His experiment used X-ray scattering instead of an OES, but I found one of his results to be very interesting and relevant to the experiments that we are doing now. He found that inter-DNA attractions began somewhere in between 1 ion per 5 base pairs and 1 ion per 4 base pairs. This is particularly interesting because the inter-DNA forces are what we are investigating, and our results should bare some resemblance to these numbers. In the afternoon, I began running the actual samples from our collaborator. I started with the first series, which is 1mM Co and ranges from 0mM Mg to 22.5 mMMg. I used a 100X dilution of these starting values though. I will analyze the results that I got tomorrow. I think that the concentrations might be a bit too low for accurate results, but they should give us a good first impression of what to expect.

June 24

On Thursday we took our final nucleosome solution and mixed a small amount of it with nuclease which ate the DNA that was between the nucleosomes. We put this mixture into a 37 degree Celsius bath and at 5 minute intervals took out a small portion and put it into a separate test tube that we then added EDTA to in order to stop all protein from working. This made sure that the nuclease would stop eating at our nucleosomes. We did this in order to determine how long we need to allow nuclease to eat at our nucleosomes to get the most single nucleosomes that we can. On Monday we are going to see how many base pairs we have in each of our timed solutions through DNA gels which when compared to a known amount of base pairs will tell us which solutions are best.

June 24th-25th

On Thursday, we continued our DNA production process. Our main goal Thursday was to conduct a trial digestion of a small sample of DNA to find the right amount of time to let it digest for. This essentially ruined the sample of DNA, but was necessary so we know how long to digest the rest. To do this, we split a small amount up into ten samples, and put in micrococcal nuclease. Then we let the samples digest, and stopped them in 5 minute intervals. When we analyze these samples, they should give us a good idea of how long to digest the rest. In the afternoon, I did some more research and worked on my presentation for Friday.

On Friday, we had our group meeting, and everyone gave presentations on their research. This went pretty well, but it took the majority of the morning. Because we had put in too many hours earlier in the week, we took the rest of the day off.

Thursday, June 24, 2010

June 23rd

Yesterday we continued the filtration of our DNA sample with additional centrifugation and stirring. We then proceeded to change the buffer of the solution by placing it in a dialysis bag with a different solution surrounding it overnight. I then ran a final test on my calibration of the spectrometer, in preparation to start testing on the actual samples today or tomorrow. I prepared three solutions of known concentration, and the results were fairly accurate, which is good. There seemed to be something wrong with the axial readings, because they were all negative, with very little intensity. We're going to do some maintenance on the spectrometer today and that should fix everything up. I also continued reading the electrostatics paper, and with Professor Andresen's help it began to make sense. I now know a bit more about the actual physics behind the competition of ions to be attracted to DNA, which is exactly what my tests will be studying as well.

June 23

Today we finished our last rinse and spin to get our the rest of the floating junk from our DNA samples. After we pi-petted out the clear leftovers we then dropped them in a dialysis tube in a Tris, Sodium, EDTA bath and left the chromatin dialyzes over night. I then continued reading my paper on Chromatin for my presentation Monday.

Tuesday, June 22, 2010

June 22nd

Today we worked on obtaining actual DNA from our red blood cell samples. This process consists of several repetitions of filtering and putting the solutions in the centrifuge. This also required the use of several different solutions that we prepared, including Tris/HCl, PMSF, EDTA and micrococcal nuclease. After essentially causing the red blood cells to explode, we used the micrococcal nuclease to eat away at the remaining substance. We controlled this reaction by controlling the temperature (putting it on ice) and by introducing EDTA to remove the calcium from solution.

June 22

Today we started the long process of separating out the nucleosomes from the red blood cells. we began by washing and centrifuging the red blood cells 4 times until basically the red had separated and we were left with a semi snotty mixture. we then cleaned the snotty nucleosomes again and collected the final solutions and combined them. We then added a mixture that broke up the nucleosomes and made them less gooey and then centrifuged again and took the liquid that was left over and tested the absorption of the leftovers and got a strange step graph where 260 nm was the middle stem. We are currently adding a salt mixture and about to do more centrifuging

June 21th

Yesterday I started the day by analyzing my results from the previous day. These results were by far the most accurate, but there were still a few issues. For some reason, when I ran the calibration series as a sample, they all were read very accurately, all less than 2% error. But for some reason my other series was still 4-8 % off. This leads me to believe that this solution wasn't prepared correctly, because the spectrometer is clearly calibrated correctly now. It would seem that spacing the calibration samples linearly and using a non-linear yields the best calibration. Later this week I will carefully prepare a new sample series just to confirm this. I also began reading another electrostatics paper that I will try and present on Friday.

Friday, June 18, 2010

Ordering and thinking

I'll let everyone reading this blog in on a little secret. A good portion of a researcher's time is spent on menial tasks and tasks that look very similar to "doing nothing". Today was one such day for me (as was much of the week). I spent most of my time ordering supplies, running around borrowing/stealing supplies from others, and planning our future projects. This last one was the most difficult, as my brain has just not been cooperating lately. People will say that this is related to the 1-month-old baby in my house, but I personally just think it's just the consequence of a more-hectic-than-normal week. To at least a little justify my presence in the lab, I poured a very dangerous powder, PMSF, into isopropyl alcohol  to make a slightly less dangerous solution. (Less dangerous because it's a lot harder to inhale a liquid than a dust.)

In real news, we are going to be doing two rather important things next week. First, we are making our nucleosomes. This is exciting, although perhaps a bit early. It's early because it is questionable whether we will have everything we need ready by that time. However, in science it is often the case that you must go ahead with plans and hope for the best. If you wait until everything seems to be ready, you never get anything done. The good news is that our Fisher rep claims our UV detector is coming on Monday. With some luck and some help from Prof. Brandauer in Health Sciences, we should be able to get that up and running on Monday. Additionally, I'm hoping to make all of the stock solutions we need on Monday so that we aren't running around trying to make those all last minute. Our collaborator, Xiangyun Qiu, is coming on Tuesday to see the prep and generally just to visit, so it this coming week should be busy.

The other exciting thing is that I'm expecting to start running the condensed DNA samples that John has been preparing the machine for. Although he's still having a hard time nailing down that last 2-3% of systematic error, I think we're pretty close to being at a place that we can run at. I'm guessing that come Friday or so, we'll be in business. As Xiangyun is the one that gave us these samples, we'll also take some time during his visit to talk to him about the samples and our general running procedure.

So it should be an exciting first week of "summer".

June 18th

Today I tried the forcing the calibration line through zero for the data set I have been working with, and I found that it was no different than the weighted linear fit. I also tried constructing an actual concentration versus reported intensity graph myself, and seeing how to best fit that curve. I found that a polynomial fit worked the best, and my result was exactly what the computer had done for its fit. Next, I designed and prepared a new calibration series, one that is equally spread over the range of concentrations we are working with. We also made a few changes to the testing method the machine uses, changing the amounts of time it is permitted to read each sample. I think that these two changes should help eliminate a lot of the systematic error we have been seeing.

June 17th

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

Thursday, June 17, 2010

june 17

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

Wednesday, June 16, 2010

June 16

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

June 16th

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

June 15

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

June 15th

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

Tuesday, June 15, 2010

We've got blood!

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

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

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

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

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

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

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

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

Monday, June 14, 2010

June 14th

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

june 14

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

Saturday, June 12, 2010

June 11th

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

Friday, June 11, 2010

1st Week Wrap-up

Well this has been a great week. Here's what we accomplished:

1. Travis ran a Mg concentrations series on some nucleosome samples. He, for the first time in Andresen lab history, saw the condensation and precipitation of nucleosomes. I don't have his data right now, but perhaps he can upload it and explain what happened. Next week he will be running the same experiment, only this time using equilibrium dialysis. This is exciting as this is the method we will eventually use for our nucleosome experiments.

2. John is really honing in on our uncertainties in our ICP-OES measurements. He's run somewhere around 30 samples total so that we have been slowly able to eliminate one possible cause after another. The last batch of samples he made before going home this afternoon should either confirm or eliminate our final couple of theories on the source of our ~5% systematic error. If he eliminates all of our explanations, then I guess it's back to the drawing board.

3. I've gotten the OK to collect chicken blood. Around 10am this Tuesday, I should be in Waynesboro, face-to-face with a pretty unhappy chicken.

And that's all for this week. Couldn't have asked for a better one. (Except for that darn UV detector that still hasn't shipped from Fisher...)

Trying to track down some chicken blood

Many of you might have wondered in the past: Where does your lab get samples from? You probably assume (correctly) that the normal procedure is to find a catalog, look up the thing you want to buy (for instance, DNA), and order it. A second method is to grow your sample, normally in bacteria, to get large amounts of sample.

For our nucleosome experiment, neither of these are practical or economical for the amounts that our group will use. So instead, we need to get nucleosomes from a source that has already done the work of making them. The preferred sources are from calf thymus (the thymus is an organ in front of the heart that is a vital part of the immune system) or chicken erythrocytes (chicken blood). I prefer the chicken blood as it is something easily collected, handled, and processed. (As an interesting side note, the blood could come from a non-mammal source. Mammal red blood cells do not have nuclei and therefore do not have significant quantities of DNA.)

Now I like to have approximately 200ml of chicken blood to create my samples. One option is to extract 1ml of blood from 200 chickens. With each chicken containing approximately 100ml of blood, they probably would barely know it. However, because it is much quicker (and as these chickens are going to be killed anyway), I prefer to collect all 100ml from each chicken.

This requires me to find a chicken butcher that allows me to collect blood from the slaughter. Normal slaughterhouses, with their incredible efficiency (for some disturbing pictures, try here) are not a viable option. So I need to find a small scale farmer that hand slaughters his or her chickens.

I was first turned on to Rettland Farms, a wonderful local farm that sells free range (really free range, unlike most labeled as such) chickens. He pointed me to his processor over in Waynesboro, PA. To make a long story short, the processor is doing relief work in Haiti, will be out of the country until August, and has someone else filling in for him. I put in a call to the cellphone of his replacement slaughterer (pictured at right), a member of a Mennonite farm collective.

So to wrap it all up, I'm waiting for a Mennonite slaughterer to check his cell phone's voice mail and return my call so I can ask him if I can hold a beaker under his chickens as he ends their lives.

Somehow we didn't cover this part of science in grad school.

Thursday, June 10, 2010

June 10th

This morning, I analyzed the results from my tests yesterday, and I found some strange results. In general, all of the predicted solution concentrations I got from the spectrometer read-outs were at least 3% larger than the concentration to which I tried to prepare the solutions. In fact, all but one set of data was over the prepared concentration. This is rather a rather disturbing systematic error, and the sources need to be identified. Though the results are not as accurate as we need, the good news is they are very precise, because if you ignore their consistant 3-6% overreading, they are then within 1%. These could be all random errors, but that seems unlikely because of their consistency. They could also being coming from my technique of solution. It could also be a systematic error coming from the pipettes. It could also be an issue with the calibration with the machine. I will investigate the source of the error more tomorrow. I did a few tests at the end of the day, and they showed that the pipettes might be cause a small portion of the error, but nowhere near all of it. I also did more research into the cobalthexamine project that I will be working on throughout the summer.
Today I increased the amount of MgCl I added to the solution in order the speed up my result process. For a few trials my solutions stayed at or below the .1 concentration mark but as time progressed my concentrations began to increase at around 48 micro liters of MgCl being added to the DNA solution. This meant that the nucleosomes that had clumped together earlier and fallen to the bottom of the solution were breaking apart and floating back up to the top. I never got my results up to a definite high concentration because as I kept taking data the Professor noticed that the amount of absorption at the 280 nm mark was very high and getting higher which according to him meant that there were excess proteins in the solution which could be the result of bacteria growing in the solution. I think the problem might have been that I started out with very small amounts at the beginning which slowed my result process and caused it to be a lengthy experiment. Also the solution that i was using were leftovers and might have just been too old. I hope to try and redo this experiment after we create our new nucleosome solutions in the following weeks and plan to use my results from this trial to influence how I go about taking data with the next experiment.

June 9th

I spent the first half of the day today organizing and analyzing the results from yesterday's samples. The results I received for the predicted concentrations of my soultions were pretty consistently 5% away from the values that I expected. This may be a problem with my preparation of the solutions, or it could be an issue with the calibration or conditions of the machine. In particular, the axial readings for the Magnesium concentrations were consistently 30%+ away from the expected value. For the most part, these results should be ignored because they are oversampled. Also, I found that consistently the first sample I tried to run was not drawn from the test tube, which led to a lot of strange negative results for one of the samples.

To try an look further into these issues, I prepared and tested a large new set of samples. This new set has two magnesium series, a cobalt series, and a phosphorus series. This should help identify where some of the 5% error is coming from. If it is a problem with the machine or the range of concentrations we are running, more data will make this easier to see. If it is a problem with my preparation, this will also become obvious. I ran the tests at the end of the day yesterday, and will analyze the data tomorrow.

Also, we are looking at changing the calibration method for the tests, because of the strange magnesium readings we were getting for the axial view. It is possible to use a non-linear fit for the calibrations, and we will see if this helps tomorrow.

Wednesday, June 9, 2010

Today was a continuation of yesterdays research I continued to add MgCl to my DNA solution and test the amount of absorption of the light we put through it. After hours of taking data i finally got to the bottom of the curve where the MgCl has pulled most of the nucleosomes to the bottom of the solution and have just started to get results of the nucleosomes freeing themselves and returning to the top of the solution. I will continue to add larger amounts of MgCl to the solution tomorrow and hopefully get the data that I am looking for.

Progress continues on a rainy Wednesday

More progress today, although not much on my part. I'll let John and Travis talk about any major results they found.

I'll tell you one thing, they've been working hard. I'm getting three times as much work done as I usually do. At this rate, we'll have all of science figured out by the end of the summer.

I, on the other hand, got a minimal amount of work done. I worked on getting Matlab installed, which is what we will use to analyze any x-ray data we end up getting. Although recent communications with the head of the beamline we were planning on using seem to suggest that this work will be later than we had hoped. Matlab can also be used to make some nicer plots of any data we want to publish. A little bit of friendly advice: Friends don't let friends publish excel graphs.

John and Travis will be happy to know that I ordered a bunch (25) of tube holders so that we aren't fighting each other for them as well as a bunch of other small things needed in the lab.

As for tomorrow, Travis should be finishing up his experiment today or early tomorrow. We'll probably pause on that so he can write up his talk that he's going to be giving on Friday. The same thing goes for John's current project. Hopefully after a day of thinking and looking at data on Thursday, we'll be going full steam ahead again on Friday.

June 8th

Today in the morning, I read through an article titled "DNA Aggregation Induced by Polyamines and Cobalthexamine". This article discussed how spermidine, spermine, and cobalthexamine could cause DNA molecules to precipitate out of solution. Then, further addition of these cations can also cause the DNA to become soluble again. The concentrations required to make the DNA precipitate are basically independent of how much DNA is in the solution. This precipitation is not caused by a classical "salting out" situation, where high salt concentrations change the activity of the water. One explanation for the precipitation and then resolubilization is that it is caused by short-range electrostatic attractions. Another explanation is that the DNA undergoes a charge reversal, but this explanation is less likely.

The rest of my day was spent preparing three different series of test solutions to test the calibration of the spectrometer. The reason for these test solutions is to find the optimal reading conditions for a later test of a specific sample that has a similar make up. I made a Mg series that was varied over 7 concentrations, a Co series over 4 concentrations, and a P series over 3 concentrations. These will be used to see how accurately the machine can read my "standardized" samples, but also I guess it will measure how well I can make solutions. At the end of the day, I was just finishing the tests, and did get to finish looking at the results. What I did see did seem to have a systematic difference of around 5%.

Tuesday, June 8, 2010

The adventures of T-money and J-pain

Today after a bit of reading on a nucleosome project I began measuring the amount of absorption of different wavelengths as the were shown through the DNA sample. For every trial i would add a small amount of MgCl to the solution and mix it up. As the amount increased there were white clouds that appeared which were the nucleosomes clumping together and falling to the bottom. My results didn't show what we were looking for and after a bit of thought the Professor determined that we needed to take a few more trials before we got the results we wanted. Tomorrow I will continue adding more MgCl to the solution and hopefully will get to the point where most of the DNA clumps to the bottom and the amount of absorption decreases and then as more MgCl is added the DNA will somehow unclump and reappear at the top of the solution.

A flurry of samples and some new collaborations being set up.

Well, we certainly had a productive day today.

To start things off, Travis started a new sub-project for the nucleosome experiments. He is looking at how the nucleosomes I made respond to an increasing amount of magnesium (a +2 ion). Theory says that as we add more magnesium the nucleosomes clump up and fall out of solution. This is an indication that they are attracting each other so strongly that they would rather be next to each other than dissolved in solution. The crazy thing about nucleosomes (and as it turns out naked DNA does this as well in different solution conditions) is that if you keep adding more magnesium, the nucleosomes go back into solution (in other words they no longer attract each other). This is pretty crazy if you think about it: You make the nucleosomes attractive by adding +2 ions and then get them to then be repulsive by adding more +2 ions. This is one of the reasons this system is so interesting to look at.

Travis started this process, slowly adding more and more +2 ions. And low and behold, we saw some aggregation happening (in the form of white wisps of material in our solution). Unfortunately, they redissolved after a bit of time.  Tomorrow we'll try to get up to higher concentrations of Mg and hopefully see the full transition.

John, meanwhile, has extended yesterday's experiments to look at a lot of samples (11 or so, I think). The reason for all of this is that we have some samples from our collaborator that we need to run. Unfortunately, when we run them, we destroy them, so we need to do it right on the first shot. To ensure that we are taking the measurements under the correct conditions, we are going to look at a lot of samples that are very similar to the ones we want to look at. Once we get the correct conditions for those samples and are really happy with how they are running, we can think about looking at the real samples. Not the most exciting thing in the world, but that's science folks: There's always a lot more build up to the experiments than there is time spent doing the experiment itself.

Finally, Xiangyun Qiu  and Chongli Yuan are joining me in a collaboration to look further into the mechanisms behind nucleosome folding. We are hoping to do and x-ray run later in the year to look at what happens to nucleosomes when you remove or modify the tails or put them in different types of solution. Our preferred tool, SAXS, should give us some idea of what's going on.

Monday, June 7, 2010

The full lab begins with a friendly competition

Well, this was an exciting day. It started out with the arrival of the full lab. Travis and John began working at 9am this morning and quickly got busy reading for their various projects while I worked on preparing samples. (For the record, Travis was given the Nucleosome Packing project while John has been assigned to the DNA Liquid Crystal experiment.) I had a heck of a time getting the calibration solutions prepared as when I was ordering I neglected to buy a funnel for pouring solutions from a centrifuge tube to a volumetric flask. A little bit of begging over at chemistry and the help of Prof. Shelli Frey and I finished the job just a few minutes into lunch.

It was important that I finished these calibration solutions as they were needed for the afternoon competition. Basically, the idea was that Travis and John would make five solutions using their best pipetting skills. This activity had two purposes: One was to give Travis and John a chance to practice said skills. Pipetting is something that is quite easy to do but takes some skill to do right. The second and more important purpose was to see how well we could all pipette solutions and how well the machine could measure the concentrations of the various solutions.

Here are the results:

Where the highlighted values are the predicted values. Everyone did pretty well. Travis knew he had a bit of an issue when all of his volumes were a bit high, but look at that precision. John seemed right on for the most part. What concerns me more, however is how poorly even the John and my values match the predicted value.

One issue is that these samples were originally meant to be diluted by 10x. We'll try that tomorrow. I'm also planning on changing the pump tubes out and trying to take more replicates. We eventually want an accuracy of ~1%.

The Adventures of T-Money

Today we took a tour of the lab and were shown most of the equipment that we would be using over the summer. We then started to read information about the projects that we would be working on. My readings focused on DNA and how the coils of DNA are wrapped around a hockey puck which is made up of two of each of the histones H2A H2B H3 H4. The histones are identical in size and fit together to form a hockey puck shape which the DNA is wrapped around twice before continuing on to another puck. This allows the long strand of DNA to be tightly compacted. At the top where the DNA comes off the puck is an H1 histone which serves as some sort of paperclip but I don'tknow the specifics of it yet. Now this configuration of DNA and histones is called the nucleosome. This is what transcription which is where some kinda genetic scanner scans a part of the DNA and then prints out a 3 piece copy of RNA which travels to proteins and fits into specific ones to activate them. I read about Transcription control which is controlling what part of the DNA gets scanned but did not read much farther into it. I also read a little about Histone Acetylation which is adding a specific acetyl group to the amino acids of the histone proteins causing the histone to loosen its grip on the DNA. We then went into the lab and practiced with the Micro Pipette creating 5 solutions of Co P Mg and de-ionized water and then testing our accuracy with the Spectrometer. The results showed that i either added a bit too much of each solution or added too much water but john later admit that he sabotaged my solutions.

June 7th

Today, after an overview and tour of the lab, we started out reading relevant research articles. The article I was reading was titled "DNA Inspired Electrostatics" by Gelbart, Bruinsma, Pincus and Parsegian. This article starts with a rough overview of DNA's characteristics and behaviors. It takes the form of a disordered coil in physiological conditions, but when polyvalent cations are introduced, it forms a tightly packed torus. Polyvalent counterions cause an attraction between charged DNA molecules that would repel under normal conditions. The electrostatics that govern these counterion-meditated attractions are still being refined, but there are two mechanisms being studied. One is a Gaussian fluctuation correction to the PB mean-field theory, which treats the counterion cloud around each rod as a one-dimensional ideal gas. It is longer range and more effective at high temps. The other mechanism is a short range electrostatic correlations between the counterions of the two clouds. This is more effective at lower temperatures. The dense cloud of counterions surrounding DNA also represents a large number of hidden degrees of freedom.

In the afternoon, we worked with preparing a few test solutions. We created 5 different solutions with varying concentrations of Magnesium. All five solutions had constant concentrations of 1mM Co and 5mM P. Solutions 1-5 had concentrations of 0mM, 10mM, 12mM, 15mM, 17mM, respectively. We also performed the appropriate calculations to prepare these solutions. After preparing the solutions with Micro Pipettes, we learned how to operate and calibrate the spectrometer. We aligned the spectrometer first, and then used three known solutions to calibrate it. Once it was calibrated, we put through the test solutions that we made. My concentration results were fairly consistent with the predicted values, and due to the fact that I sabotaged Travis's results, they were precise but rather inaccurate.