Thursday, August 5, 2010

August 5th

Today I put the finishing touches on my presentation. I added a few graphs from the first series, and changed around a bunch of the slides. Other than that I just worked on an outline for my own benefit. After listening to Alex's presentation, I gave mine and received criticism on it. I still have some work to do on it, but it should be ready for my presentation in the spring.

August 4th

Wednesday, I spent pretty much the entire day working on my presentation. I have made a series of animated pictures that will help explain overcharging and the counter ion density wave theories. I also recruited the aid of Atwater Animations Inc. to help with some of the method slides. I think that over all, my presentation is really coming together.

August 3rd

On Tuesday, I finished up with my final analysis of the data for the second series. I calculated all of the averages a different way, one which I found to be a much better representation of the data. I got the concentrated series to agree very closely with the diluted trials, which is very promising. I also began updating my presentation for Thursday.

August 2nd

I spent most of Monday organizing and analyzing the data from the second series. This included looking through all of the raw data from all of the trials, and making sure there were no large problems with it. I also updated it to take into account the standard deviation of the mean.

Tuesday, August 3, 2010

Monday, August 2, 2010

July 29

Today I ran 2 digestions with the only difference being how much nuclease i put in each sample. In one i put a 10x dilution of our nuclease and in the other i put a 100x dilution. I kept everything else the same and used our old protocols to complete the digestion. On Monday we are going to run the gel because we have been kicked out of the chemistry department for the rest of the week.

July 29th

On Thursday, I made some new spreadsheets to analyze the data in a different way. I am trying to average the data in different ways, by averaging the values across the data series first. I think that doing this way is more accurate, because the trials are fairly consistent, and this helps to take into account where most of the error is coming from. This process seems to be making the data agree with itself more, which is promising.

July 28th

On Wednesday, I finished running the concentrated trials of the second series. I also reprocessed a few of the earlier trials, trying different calibrations. I discovered and correct one major problem, which was that I had run out of sample in one of the trials. This was throwing that trial way off, because it was taking values when there was no sample left. Reprocessing some of the data with different calibrations also helped make the data more consistent.

July 27th

On Tuesday, I continued to prepare and run the concentrated trials for the second series. So far things are going well, but the data is not quite matching up with the diluted trials. This could be because of the way I am analyzing the data, or also from problems and inconsistencies in the data itself.

Thursday, July 29, 2010

Many general lab updates

For our nucleosome project, we have had some setbacks, most of which we have recovered from. As you remember from last time, our column had broken. That is still the case and we are still waiting for a replacement as it is on back-order. What I had failed to mention was that the sample itself had also had some issues. Namely, there was a white powder that had precipitated out of the sample and to the bottom of the tube. This was disturbing as I was relatively sure that this was all of my nucleosomes aggregating and falling to the bottom of my tube (something they are only supposed to do when I want them to). Since we were stuck without column, I thought I would investigate this issue.

It turns out that nearly all of my nucleosomes had aggregated. After some investigation, it turns out that the nucleosome sample was actually in 10mM Calcium Chloride (a +2 ion) instead of 1mM. This is an issue because nucleosomes aggregate at about 5mM of +2 ions. It is also a problem because the EDTA we add to stop the digestion is added to stop 1mM of CaCl2, not 10mM.

The first thing I needed to do was get the nucleosomes back in solution. We did this by dialyzing the nucleosome solution (and the precipitate) with 150mM NaCl (a +1 ion). This is done by putting the nucleosome solution in a bag that only lets ions and water pass through it, but not the nucleosomes. It turns out, if you add enough +1 ions, they will displace the +2 ions at which point the nucleosome should go back into solution. After a day of dialysis, we had significantly increased the amount of nucleosomes in our solution. After a weekend of dialysis, we had gotten back basically all of our nucleosomes. Now we had to make sure that they had been digested correctly (especially checking for over-digestion which shows up as smearing on our gel).

The results look like this. Now the furthest column to the right is our DNA ladder. This is used to let us know how long our DNA is. You match the known length of the ladder band to your sample. The bright band at the bottom is a 100 base pair DNA. Each step "up the ladder" is a 10 base pair step (110 base pair, 120 base pair, etc). The lane immediately to the left of the ladder is our sample. If you look and count carefully, you can see that it lies between the 140 base pair and 150 base pair "steps" of the ladder. This is perfect as the DNA should be 146 base pairs! Even better, there is no "smearing" to lower parts of the gel, which would indicate over digestion. You can see an example of this in a different sample that was run in the lane immediately to the left of our sample.

So as a wrap-up, we have the same amount of nucleosome we started with, all in solution and digested to the correct length. All that is left to do is to separate out any small amounts of double or triple or larger nucleosome arrays and we will be done. Since we are still waiting for our column to arrive, we may end up doing this through a sucrose gradient instead.

Not to be shown up, John is busy plowing through the ton of data that he has taken. Now that we have all of the data, we need to figure out why some of it looks great and some of it looks not-so-great. Things looked pretty bad at the beginning of this process, but each time I check in with him, he seems to have found another correction to make the data fall in line. If all goes well, we'll have it all whipped into shape by the end of this week.

Tuesday, July 27, 2010

July 27

Today I ran tests on the Lambda 25 to see if it is properly working, and it passed all the tests I could do without having to make any samples. I also created an instruction sheet for the machine with the basics so that anyone can now use it.

Monday, July 26, 2010

July 26th

Today I made and ran a second series of concentrated samples for the first series. The results came out somewhere in between the diluted series and the first concentrated series that I ran. This is promising, because the results should all be the same. It is possible that there is some sort of concentration dependent interference that is happening, which could be causing some of the differences. Today, I decided to start running concentrated trials of the second series, and started preparing the samples. This should be easier because there is much more of the second series left. Overall, I am finding that the eighth and tenth points are still uncharacteristically high, which leaves me to believe it is something in the actual sample preparation. This is one of the only explanations I can see left, because I have ruled out most of the possible problems on the testing end. One possible explanation is that not all of the Mg buffer solution was removed when the DNA precipitated.

July 23rd

Today, I wrote the second of two results reports, and updated all the graphs for the second series. It was Matt and Andrew's last days, so we had their final presentations today as well.

July 22nd

On Thursday, I began by analyzing the concentrated trial for the first series. It came out to be significantly lower than the eight diluted trials I had run previously. By lower, I mean the the normalized concentrations for the concentrated trial should have been the same as the diluted trials, but they were consistently below the diluted trials. This was kind of disturbing, and has prompted me to run more concentrated trials. I also updated my results report, and fixed and updated all of the graphs that I have been creating. I also consolidated and organized all of my data and graphs.

Thursday, July 22, 2010

July 21st

Yesterday, I started by running the last trial of the second series. I also prepared a few more tests and ran them as well. The first was a new test for chlorine, which worked slightly better than the first. Part of the problem was that I didn't prepare the NaCl standard quite right, but some of the results still come out negative. I also ran some of the Chromatin that the professor and Travis have been working on, and found that it had higher calcium concentrations than they expected. I also designed and ran a highly concentrated series of the first series. This should help identify what is actually going on in the last few samples of each trial. They have been fairly random, but almost always are way above where I would think they should be. This is attributable to the fact that are very low concentrations of all of the elements. This makes sense, because less DNA is precipitating.

July 20th

On Tuesday, I continued to run the second series of samples, and I finished all but one of the eight trials. I also began to look at the spectrometer's ability to analyze chlorine in samples. This will be useful because it is possible that the cobalt hexamine ions are not completely dissociating. What this means, is that some of the cobalt ions we would expect to be +3 will actually be behaving as +2. Knowing the exact concentration of Cl would allow us to see if all of the ions were dissociating. Unfortunately, my first results came out very strange, with the chlorine concentrations going negative for a lot of the values.

July 21

Yesterday I spent the morning looking through the spectrometers manuals trying to figure out why the results we are getting are so weird. While doing and alignment i put our sample container into its slot and noticed that only a small amount of light was going through the sample compartment and the rest was just going over it. So the professor re-ajusted the holder and I ran another trial on matt and andrews red and purple solutions and got better results than the first time. I also re-ran the collaborators chromatin and got its absorbance to be 3 times what it was when i ran it the first time.

Tuesday, July 20, 2010

July 19th

Yesterday, I continued the testing on the second series of samples. I prepared three more trials, and tested four of them. I also began analyzing the first few trials that I have run. I've found that the ends of the series, near 45mM Mg have final concentrations that are very low, near 1-2 ppb, which is very hard to measure. While this results can still be used, it is possible that they are inaccurate. I also started correcting and adding more graphs to my report. I am adding some total charge and final concentration graphs. Due to the low concentrations, I am going to look at designing a concentrated trial today.

Friday, July 16, 2010

July 16th

Today I prepared the next three trials for the second series, and ran two of them. Everything is going well so far. One thing that I am considering is running a trial at a higher concentration. I think it would be interesting to see if only diluting the solution to 50X or 20X had any effect on the results. I think part of the reason that the results are going so wacky for the higher concentrations of initial magnesium is that the precipitated DNA concentration is so low. I think that running it at a higher concentration would help get accurate data for the higher points. Unfortunately, we don't have enough sample right now to do this several times, so I will have to plan it carefully to conserve sample. We also had our meeting updating everyone on progress, and also describing some of the work we still need to do.

July 15th

Yesterday, I ran the first trial of the next series. This series goes from 0 mM to 45 mM initial Mg concentration. As I looked at the results from this first trial, I was glad to see it closely resembled the appropriate part of the first set of trials, which went up to 22.5 mM. The last few points, towards the higher concentrations, and further trials should help see what is going on there. I also reviewed my results report for the first series, and updated all of the graphs. Unfortunately, we found an error in how I was calculating the errors in the measurements. Due to the way I uses excel spreadsheets repeatedly, this error was in all of the data I have analyzed so far. I was able to track down all of the repeated mistakes, and I believe my analysis is no more accurate.

Past few days

Basically in a nut shell we did another trial digestion using .1 microliters of nuclease instead of 1 microliter and we got the best results to date. We took our data and determined that we would get the best samples with a 20 minute digestion so we did a digestion on the rest of our chromatin. We set up our size separator in the cold room and as we were pouring in the chromatin the professor noticed that our tube had dried out making it useless, so we quickly poured our DNA out and lost a fair bit and then took the tube back to the lab to clean it out. However, during the cleaning we didn't use a Styrofoam sleeve to protect the glass and accidentally over tightened and broke our tube, so now we are at a stand still waiting for new materials. With my free time i have worked a lot on the website and did quite a fair bit of reading. Today I took the scatchard plot of the nucleosomes from a similar experiment to ours and used a program to scale and choose each of the points to figure out what their actual locations are and then turned it into a read-able graph that we will look at on Monday.

Thursday, July 15, 2010

Great Successes and Amazing Failure

A lot has happened since I last posted. Let's start with the positive.

We have successfully run a very nice non-denaturing poly-acrylamide gel. The key was to increase the amount of sucrose in or loading solutions. This helped minimize diffusion of the samples before they started running. Another thing that helped was a mistake I made. In my haste to make the gel, I accidentally inserted a 10-well comb rather than a 15-well comb. However, this actually resulted in much nicer results.

We see exactly what we would like. The first column is our normal DNA ladder. The second is the undigested chromatin. As you can see, it is all at the top of the gel. Then we start digesting. As we go to the right, the time of digestion is increasing. As you can see, by the sixth column or so, the size doesn't change much for a few columns. This is the digestion being stopped right at the nucleosome core. After a bit of time, however, the digestion proceeds past this point in some of the nucleosomes, digesting the DNA that is around the core. That is why the gels get smeared towards the bottom for the last three columns. When we do our final digestion, we will make sure that we don't let it digest this long.
After this great success, we digested one of our samples and were going to pass it through our size-exclusion chromatography column. This column will separate our sample by size. This will allow us to collect all of the nucleosomes that are the exactly correct size, while ignoring those too big or anything that is too small (bits of DNA, etc.). The setup is shown to the right.

One thing about a size-exclusion column is that it must never dry out. The tricky thing is that you need it to almost dry out to add your sample so that your sample stays relatively concentrated in the gel. To make a long story short, I brought the column to the almost dry level and the closed the valve. However, the valve leaked slightly and after 15 minutes, when we loaded the sample, I noticed the gel had completely dried out.

At this point we needed to recover the sample (which we did right away). We then took the column back to the lab to recover the media (the white stuff in the column) so we could rehydrate and repack the column. During this process, I overtightened the column in the vise that was holding it. So now we are waiting on a replacement column and replacement media. It's funny how much a small leaky valve and 15 minutes can change things.

Wednesday, July 14, 2010

July 14th

Today, I had the Spectrometer running the entire day, and I made it through all 4 trials that I wanted to run. So far, I have found all of the results to be fairly consistent with my first set of four trials, which is good news. I think that the new calibration standard may have helped to lessen the extreme errors I was getting with the eighth and tenth points. All together, I now have 8 trials that I can include in my report of my results so far. This should give us a very good idea of the patterns of the ion concentrations. Tomorrow, I am going to start preparing the second series, which is also a magnesium series. The second series goes from 0 to 45 mM Mg. I will also being finishing up my report of my work so far, which I will probably be putting up on here. Travis and I have been putting together a website that summarizes the work that we are doing, which can be seen here: This will be a good overall explanation of the theories and processes involved.

July 13th

Yesterday, I analyzed the final of four initial sample tests that I have been running. It came out to be similar to the other three trails, which is good for consistency. I will now be moving on to the next set of trials for this first set of samples, which I expect to be even more accurate. Using a better calibration standard should make this set of data more reliable. I also tested my new calibration standards, and found that they worked quite well. After diluting each one to the proper concentration, I am left with about 80 test tubes just waiting to be tested, which I should hopefully be able to finish tomorrow.

Tuesday, July 13, 2010

July 12th

Yesterday morning, I read two different articles. One was about DNA stretching, and the other was about incomplete ion dissociation. The DNA stretching article was not as relevant as I had hoped, but it was still interesting. It was about a statistical mechanical model for the condensing of DNA under tension. It was based on data similar to that which I am collecting, on the condensing of DNA in the presence of Cohex. It gave approximate values for the tension present when DNA collapses. The second article provided another theory as to why the DNA would condense and the resolubize under increasing amounts of Cohex. It suggests that as the concentration increases, not all of the Cl- leaves the Cohex, so there are increasing concentrations of Cohex+2 instead of the normal +3. This would mean that the Cohex is actually out-competing itself, causing the DNA to return to solution. It is possible that our results could be supporting this theory, although it is hard to tell without the Cl- concentration. This is unfortunately impossible to get because the solution was flooded with NaCl to resolubize the DNA. In the afternoon, the nitrogen finally got here, and I was able to resume testing. The first thing that I tested was the fourth of four initial runs that I did on the real sample.

Monday, July 12, 2010

July 9

On Friday I spent the day preparing another trial digestion this time using .1 micro liters of nuclease instead of 1 micro liter. Because our previous gels showed our digestion ending very quickly we decided that we were adding too much nuclease making the whole process end after around 5 minutes. So I did another 50 minute digestion and made a 10X dilution. I also did a digestion on the collaborators nucleosomes using the same method. For these samples we also added a lot more sucrose than before also in higher concentration. The professor hopes that this will make our gels much more readable then they have been.

Friday, July 9, 2010

July 9th

Today, I spent pretty much all day making new samples to test. I was anticipating getting the new nitrogen tank today, which would let me run some of these samples, but it hasn't come yet. I have two new calibration standards, which are both designed to span the exact range I'll be working with. They are both the same thing, but prepared twice just to make sure that sample preparation doesn't have anything to do with the results. I also prepared these calibration standards to be tested for accuracy as well, by separating them into additional tubes. I also diluted the DNA samples into 4 different sets, each of which are 100X dilutions. Today, the professor gave me a paper that has possible applications to our project. The paper suggest that some of the Cohex did not completely dissociate in the original solution, making some of them +2 ions. This would help explain why our results are significantly higher than the existing PB theory would suggest.

July 8th

Yesterday, I worked on analyzing the data in a few more different ways. I separated the data so that it showed just the average Mg, Co and P concentration. This helped me to explain why two of the points don't fit in my contributed charge graphs. This is a combined effect from the Mg and Co values being higher and the overall DNA (P) concentration was lower. I also started to design and make the next huge set of samples that I will be testing when we get more nitrogen.

Thursday, July 8, 2010

And more DNA gels

Well, we keep getting better at least. This digestion showed a much better digestion. As you can see, the 0 minute digestion showed a smear of DNA, while all lanes that were digested for 5 minutes or more showed a nice single band that can be believed to be around 146bp. As can be seen we have a few issues.

First, the digestion is supposed to take around 30 minutes. This is helpful because it means that you can make sure that you are just digesting to 146bp and not overdigesting. Ours is taking 5 minutes. In our next digestion, we'll try decreasing the amount of nuclease (the thing that "eats" the DNA) by a factor of 10. Hopefully we'll then see a little more of a progression and less of a step function.

The second issue is that the gel still doesn't look great. The first issue is the ladder. We obviously didn't run long enough: Our 100bp standard only went about 1/2-way down the gel. However, conditions were controlled enough the last time that we should be able to have a nearly-perfectly timed run.

The other issue with the gel is that the DNA in all but the ladder lane is quite diffuse. It would be much easier to read if they looked like lines, similar to the ladder in previous gels. My theory is that this is due to the sample not being dense enough when loaded and therefore diffusing before it starts running in the gel. An easy fix for this will be to increase the amount of sucrose before loading these samples.

Finally, a problem with the gels that I'm not showing you is that I messed up the loading of the other gel. This is actually Travis's gel. Mine didn't have a ladder! Well, I'll have to do better next time.

Things with John's project are going quite well as you can see by his posts. We should have nitrogen tomorrow which will allow him to run more samples. After that starts up, we should be on our way to having all of the DNA aggregation samples done by the end of next week if everything goes well.

July 7th

Yesterday I spent most of the day reformatting the data that I have already collected. Instead of comparing the results across the different trials that I have run, I compared the results across the different collection methods the spectrometer uses. This gave me three more sets to compare, the Radial view and the two Axial views. This took a lot of manipulating the data, because everything I have done prior has been set up to compare different sets. Fortunately though, the end result was that I confirmed all three methods are equally valid. This means that I can keep using all three sets of data without fear of one of them being massively inaccurate.

Wednesday, July 7, 2010

July 7

Today we poured and ran our DNA gels.. the professor put in the concentrated samples and I put in our 10X dilution samples. To get the samples in the gel we had to take a pipette and slowly inject the solution into the wells in the DNA gels... After a while this gets difficult because you really have no room to mess up because if you go side to side you can mess up the wells and if you go to far down you can puncture the gel. But I finally got the hang of it and after some time we were able start our gel at 10V. The professor decided to use a power supply from the physics department because last time when we used the power supply that is in the lab it shut down during the night and messed up our results.

July 6

Today I ran another timed digestion going up to the usual 50 minutes. I created a 10x dilution of each sample and also made a sample of the old nucleosomes we had in the freezer by adding just enough to make the concentrations of the new nucleosomes samples and the old sample equal. tomorrow we are going to run the DNA gel which will probably run overnight.

Tuesday, July 6, 2010

July 6th

Unfortunately, the spectrometer ran out of nitrogen over the long weekend and we won't be able to replace it for a few days. This means I have to put off testing for a few days, but it also gives me time to look at the results that I have so far and plan the next set of tests. Today I worked on finding the uncertainties for all of my data and seeing how they compare to the theoretical Poisson-Boltzmann prediction. A few things bother me about the final fit that I came up with, which is seen above. First of all, PB theory would suggest that there should never be more than 1 total charge being compensated for, which there often is. Second, the eighth and tenth data points are disturbingly large. Third, the error bars for only the eighth and tenth points can be seen on the graph, meaning they are by far the largest errors. Fourth, the PB predicted curves shown on the graph as "Theoretical" had to be translated to fit that well on the graph. The Cohex curve had to be multiplied by a factor of 1.25, and the Mg curve had to be multiplied by a factor of 1.6. This means that my results are systematically higher than they "should" theoretically be. Also, I believe that once the new nitrogen comes and some maintenance is done to machine, combined with the new calibration standards I will make tomorrow, the results be more precise. This should help identify where the problems are coming from. Also, I should say that the third trial I ran, which is identical to the fourth I have yet to run, was prepared slightly differently and has already shown to be more accurate.

Friday, July 2, 2010

July 2nd

I spent most of today analyzing and writing up my results from the past few days. Some good news, is that the data sets are fairly consistent with each other. I graphed the charge that was contributed by both ions to compensating for the DNAs negative charge, and it actually appears to have a somewhat linear relationship. There are two points that do not fit the linear fit, the 8th point and the 10th. The 8th is somewhat disturbing, because it appears to be random. I originally thought I might have prepared the solution poorly, but now that I have run another differently prepared solution, I find that hard to believe. Who knows though, it is very possible it should be like that. The graph on the left is the average of the 3 data series that I have collected so far. It is also a little strange that there are often more than 1 total charge contributed by the sum of the Mg and Cohex, because DNA only has 1 negative charge per phosphate. Because the Mg and Cohex are normalized with the P concentration, there should never be more that 1 charge total. This suggests either some error, or some sort of overcharging. I am nearly done with the analysis paper I have been writing, and will most likely post it on here when it is finished. Next week I will be finishing analyzing this data, preparing the new calibration standard, and running as much more sample as possible.

July 1-2

Thursday was a pretty relaxed day as we waited for our DNA gels that shutdown over night to finish up. While we waited I read an paper on how Magnesium affects chromatin but it was a little dense and hard to understand so I'm still making my way through it. When we finally got our DNA gels and pictures of them we found that we had let them run too long and that the data we were looking for had run off the gels. But we had better looking gel than our first try so we are narrowing down our results and should get it the next time. Today I re-tested the old nucleosome samples to determine which one that we had was the best sample compared to our new DNA. after some confusion with the results I finally got the data and determined how much nucleosomes per gram are in the solution and how much we need in order to have a good comparison with our new DNA for the next time we run a gel.

New Gels, New Challenges

Note: I realize these posts may be confusing to those outside of biology/biochemistry fields. In general, gel electrophoresis separates molecules by size: In this case, biggest are at the top and smallest are at the bottom. For more, look at this great animated introduction.

As you remember from my last post, our last gel ran quite well, but the results showed that we had failed to digest our inter-nucleosome DNA. This time, we modified our digestion process (and by modify, I mean went back to the process I had been using before I decided to "improve" my process).

This time however, we have another issue. The gel itself gave us problems. There were a couple issues layered on top of one another, but the main one was that our power supply that runs the gel decided to quit some time in the middle of the night. This had two effects, one was that the DNA had time to diffuse and migrate in any old direction it wanted to during the time that the power supply was off. This causes a broadening of the bands which I think we are seeing here. Another issue was that it forced us, due to time considerations, to ramp up the voltage to very high values which I think is what caused the intense upside-down V-like shapes to the bottom of our gels. Finally, we ran the gel a little too long losing our 100bp marker. In the previous post, you could see the 100bp and 330bp markers, but I am pretty sure that the marker you see in the first lane is simply the 330bp marker. This makes it impossible to determine as, even though some of the middle markers are resolved, we have no way of counting up from the markers (each sub marker is 10bp).

As for the digestion itself, things look pretty good. We got rid of our smear of DNA and it is obvious that the DNA is migrating to approximately the correct place. There are only two issues I am worried about. One is that aside from the zero time point (the second lane), most of the lanes look the same. In other words, it seems as if we are completely digested by 5 minutes.

The second issue is that the lanes that do look different are completely random. The corresponding times are 20, 40, and 50 minutes. By 50 minutes, we shouldn't see anything above that band of brightness on the bottom. The fact that it is random seems to indicate a possible pipetting error. However, I ran and loaded two separate gels, so the pipetting error would have had to have come earlier. We'll try going over our process to make sure that this isn't an issue.

Other than that, it's back to do the exact same thing again. This time we'll be using a different power supply so we don't run into the same problems.

July 1st

Yesterday I began analyzing my results from the previous day's testing. The results seem to show a clear pattern, for as more Mg is added to the initial solution, more Mg is precipitated, suggesting it out competes the Cohex. In general though, as more Mg was added, less DNA is found in solution, suggesting that Mg does not precipitate the DNA nearly as well. I also began working on a report that shows my process and an overview of my results so far. I also prepared another set of samples that should be exactly the same as the ones I ran the last few days. Running these samples again should give me a data set large enough to get some good statistics for my results.

Thursday, July 1, 2010

June 30

Today I spent the day making new timed digestion samples that we could run in the DNA gels. After I finished the final bath that the samples take in 50 celcius I created 10X solutions with each of the time trials because last time we made a gel it was very bright which is an indication that there is too much DNA. We ran the DNA gels overnight and tomorrow we are going to dye them and see our results

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.

Monday, April 26, 2010

Making mistakes allows time for posting to the blog

I want this blog to be an informal collection of information related to my research lab and so I'll start it about as informally as possible.

The reason I am able to post right now is because I have made a large mistake with the machine. I failed to connect the peristaltic pump tube correctly (specifically the outlet, or red, tube). This caused the spray chamber to be filled with liquid which eventually (thankfully) extinguished the plasma. I was in the room the entire time, ignoring everything that was going wrong. I will have to see if I can set the software to make a loud sound when things go wrong.

To recover, I started the pump back up on "flush" mode (which is simply a fast pumping mode) and waited for it to drain. When it finished draining, I started the plasma back up. The plasma extinguished quickly after igniting, but I was able to reignite it and it has (as of writing) been lit for about 10 minutes. When 30 minutes has passed, I'll start to align the plasma, calibrate, and run my new "Mix" series of samples.

(Note: Relevant page of notebook is 35.)