Week 6 & 7 - DNA Wrapping & Starting Over

During the beginning of week 6 and 7, I continued DNA wrapping. I tried using different concentrations of each the DNA and AuNPs, however, nothing worked well. I could not replicate my very first try, which, although was not useable due to excessive aggregation, still was indicative of wrapping due to the surface charge flip from positive to negative. After trying to wrap with this sample multiple times, I concluded that these particles were simply not useable for wrapping even though the measurements were not bad. To confirm my suspicions, I still had a little bit of the pellet I used for my first attempt at wrapping, so I tried it again with that. As expected, the surface charge flipped, indicative of some wrapping. So I threw my new samples out.

Attempting to start again with wrapping, I noticed I had old citrate particles that were very good lying around. I didn't want to waste good particles, so I tried with these. It ended up being a waste of time. I coated a bunch of them with polymer, which worked out very well, and cleaned them and pelleted them. These looked much better than the other samples that I had been using.

These are old particles from the beginning of research that looked very promising under the UV-Vis. As you can see, every step is not aggregated at all and is only widening a little bit. I was hopeful that these might work.

After calculating the concentration using the method I used before, I diluted the pellet 10x in order to keep them from being too concentrated. The pellet ended up being at least 2x more concentrated than the previous pellets, so I didn't have to waste as much in order to make the same amount of solution.

After wrapping, zeta potential showed that the surface charge was still positive. However, it was only slightly positive at around 12mV. This is indicative of slight wrapping, but the majority of particles were still not being wrapped. I concluded that this was most likely due to the sample not being cleaned enough, and there was simply too much PAH floating around that the DNA could stick to instead of the particles. So I tried to clean my samples again to see if that would help. Unfortunately, this completely destroyed them in the process. The data was extremely weird, the sample looked extremely polydisperse and they had probably the most horrible looking absorption spectrum I've ever seen. However, the surface charge was 0mV. From this, I decided no more. I was going to figure out how to clean the particles and remove excess polymer without aggregating them. So I looked through a bunch of different papers.

Moving on to week 7, I had looked through a lot of different papers that coated AuNPs with all different kinds of polymers. A lot of the papers said that they centrifuged the particles at high speeds of 9000-12000rpm for a short amount of time (5 - 15min). I was surprised at this but decided to give it a try. I ended up going with the same speed and time that the people in the chem department used in order to coat with polymer, 9000 rpm for 15 minutes.

I also noticed something in the papers. Almost all of them had some form of saying, "in order to keep samples as good as possible, we always used freshly made particles for our experiments". I realized that maybe freshly made particles are just better to use than old ones, for some odd reason. It's probably because it's super hard to make perfectly stable particles, so they're always going to worsen a little bit over time. So I made new particles, batch 4, and went through all the steps. They looked very promising every step of the way.

Next week I'll start making DNA-AuNPs using my new particles.

Weeks 4 & 5 - Nanoparticle Pellet Preparation & DNA Wrapping

During the beginning of these weeks, I focused on perfecting the polymer coating procedure from before. I got a few good attempts, in which the data looks decent enough to move on to DNA wrapping, but overall, there is no deciding procedure that works the best. This is somewhat disappointing; when I need to make new PAH-Cit AuNPs in the future, I have to either get lucky or find a way that does work most of the time. For now, however, I am going to use the ones that are decent enough to move forward. These are all from the same batch but are separated into 15mL conical tubes in order to make the centrifugation go smoothly. Their absorption graphs look decent, with only a small shoulder which indicates some aggregation of particles. This isn't too big of an issue, though, so they are cleared to wrap with DNA.

In order to get the particles ready for DNA wrapping, I needed to make the concentration higher. The concentrations were extremely low which would not work very well. To do this, I centrifuged them at 4000rpm for 45 minutes, removed the supernatant on top, combined the pellets and recentrifuged the supernatant until no pellet emerged. This would eventually give me a concentrated sample of NPs. 

I also needed to prep the DNA for wrapping. To do this, I made a sample of DNA dissolved in TRIS buffer that has a steady PH. Once dissolved, I sheared the sample with a probe sonicator. This is a technique that essentially shortens the DNA to a specific length that allows for wrapping. The machine makes a really loud noise, so I had to wear ear protection. After this, I realized I used the wrong concentration of TRIS buffer before, so I had to dilute it in ultrapure water (deionized to ensure the DNA's charge was not messed with) and a certain concentration of NaCl, which is needed later in the process to ensure the DNA and polymer are all surrounded with charges, but we decided to add it in then to save time. I also measured the absorption spectrum of both the sheared and unsheared DNA samples, in order to obtain their concentrations. This didn't turn out well.

Sheared DNA VS Unsheared DNA UV-Vis spectroscopy. The peak should be at 260nm, but it's much higher. I'm not really sure why this is, although it might have to do with the machine not being calibrated correctly or my TRIS concentration being too high. 

Once the sheared DNA had been buffer exchanged, I ran another UV-Vis characterization to see if they were any better. I then realized I had been using a plastic cuvette to measure the spectrum, which, if you know anything about physics, is impenetrable by UV rays. Since the wavelength parameters are set to numbers in the ultraviolet range, I wasn't actually getting real data. So, I re-ran the measurement with a quartz one, and they turned out good, with peaks of about 260nm (expected).

The absorption value at the peak, 0.13587, enabled me to calculate the concentration of DNA in the solution, which turned out to be around 0.68 mg/mL. For wrapping, I need to dilute this by about 10x.

Meanwhile, I finished pelleting my particles and needed to calculate their concentration with the UV-Vis as well in order to start wrapping.

Extremely concentrated gold nanoparticles. This is only about 2mL, as each time the pellets are combined it only adds a few μL of particles. These were already cleaned with the centrifuge process before pelleting.

When I ran the UV-Vis I had to dilute the pellet 100x. The particles looked decent. Some mild aggregation and thickening of the peak had occurred, but I deemed this not significant enough to not continue wrapping. In python, I programmed the files from the machine in order to set up a graph, as usual. However, this time, in order to clean the samples, I needed to separate them into smaller tubes, 8 to be exact. There were 8 samples that looked extremely similar to one another and were going to be recombined anyway. To get the picture that you see below, I attempted to take the mean of the absorption values of all 8 samples at each wavelength, normalize them, and plot them again as a new line. This took me two hours, but it was worth it, as now the graph is much cleaner looking and easier to understand.

The original PAH-Cit AuNPs cleaned twice with a centrifuge versus the pellet solution (a dilute version), normalized. As you can see, the pellet has a thicker peak and a slightly bigger shoulder.

With this data, I used the Wolfgang Haiss protocol from another academic paper in order to determine the concentration of the particles. The procedure is as follows: take the absorption value at the highest peak, divide it by the absorption at 450nm, use the table to determine the diameter, and then use another table to determine the molar decadic extinction coefficient, which can then be used in the equation c = A450/ε450 in order to determine the concentration in moles. All of the calculations have been done in the paper, so I simply had to follow the protocol. When I did this, I finally got a concentration of 39nM for my pellet. I then diluted it by 10 in order to not overwhelm the DNA.

The first ingredient for the final mixture, diluted pellet from my PAH AuNPs.

                                                

I diluted the DNA 10x simply by combining 1mL with the particles instead of diluting it with water and then adding 10mL, which is essential because I don't want the water to contaminate the concentrations of NaCl and TRIS that the DNA is combined with. This gives me a final concentration of 0.068mg/mL DNA.

The second ingredient for the final mixture, diluted sheared DNA.

This is my first attempt at creating DNA-PAH-Cit coated AuNPs. I shook it vigorously in order to make the DNA wrap around the particles, which Dr. Andresen said might work because we really have no idea what we're doing and we have to try something. I then left it overnight.

The next morning, they were a little purple in color. I ran UV-Vis measurements as well as DLS and Zeta potential. The absorption spectrum looked worse than the pellet, which I guess is to be expected, but since the pellet was already kind of aggregated, it made it worse.

My DNA wrapped particles compared to the original cleaned and pelleted polymer particles. The black line, indicative of the DNA wrapping, is aggregated. Ugh.

                       

I still wasn't completely sure if this was terrible or not, so I began to clean the wrapped particles 2x in the centrifuge and run the tests again to see if they were better. After cleaning, I noticed that the tube had a clump of aggregated particles at the bottom and I immediately knew they weren't going to be useable, but I still ran them through the UV-Vis, confirming my doubts. So much for the first attempt.

I decided to redo wrapping, but with a different set of particles. Luckily, I had other even better PAH ones that I had been pelleting along with the others and eventually worked my way up to 1mL of pellet. I then checked their absorption spectrum to ensure these were still as good as they were before. These particles were so much better it was insane. There was almost no shouldering even on the pellet solution, as opposed to my first try. 

Pelleted particles compared to original citrate and PAH-coated particles. As indicated by the black line, there is almost no aggregation and only slight broadening of the peak that has occurred. This has potential.

I knew I had to save as much of this pellet as possible and not waste it with possible aggregation with DNA each attempt. I decided to dilute the pellet 50x instead of 10x in order to save the particles for as many tries as possible. I made 3 samples of the diluted pellet because the pellet has a greater chance of aggregating on its own if let sit out, simply because the particles are more concentrated and closer together. I only used 200uL for each dilution, which is great. I calculated the concentration using the method from before, and then I mixed the two ingredients together, 1mL sheared DNA from before and 9mL of the diluted pellet, in order to make 10mL of the DNA wrapped particles. Instead of shaking it vigorously this time, I decided to invert the tube a few times and let it sit a while instead. I noticed that I did the same thing when coating with PAH, and even when I made the original citrate particles. I didn't disturb them at all, I just mixed them a little and then let them sit. I was hopeful that this would not cause aggregation but would still allow for the DNA to wrap around the particles.

My DNA-PAH-Cit AuNPs attempt 2. As you can see, they are a nice pink color, as opposed to the purplish color that arose from the previous try. These should be better.

I then let them sit for a few hours and ran UV-Vis and DLS/Zeta on them. They seemed a little weird - the UV-Vis looked somehow unchanged from the dilute pellet, which was strange because I was expecting at least a little aggregation. The zeta potential was also weird because the surface charge was about 40mV. I was expecting this number to be negative since DNA is negatively charged, so I guess wrapping didn't occur. The pellet's charge was about 55mV, so I guess it did reduce it a little, but not nearly enough. It should be around -30mV. I deduced two things from this. Either the wrapping didn't occur because I didn't shake it enough (maybe the particles are stubborn), or I didn't add a high enough concentration of DNA, so not enough stuck to the particles. I'm not sure what concentration to do next, or if I should stick with the original and try to shake it a little more. I'll do that next week.

Finally, I started pelleting my other particles that were good, even though they are only 10mL each. Next week, I'll finish pelleting these for later on, as well as make a new batch of DNA wrapped particles probably using a different concentration. I'm just happy that I was able to progress to the final step of preparation in my research. After I get these and they look good, I can begin experimentation. Hopefully, they work out!

Update on preparation of nucleosomes

I am about 4 weeks into research (half way done with my research here on campus for the summer) and so close to having mononucleosomes. Since the last time I posted, we have made progress on preparing nucleosomes. The past couple of weeks have consisted of digestion processes with the DNA and running gels to try to figure out if we had nucleosomes in our samples.

Figure 1: Column Trace of NCP (nucleosome core particle) prep samples

Last week, Dr. Andresen ran a column with the samples we have of DNA. Figure 1 shows the results of the column which helps us determine what each sample has. Each green number indicates a sample number. Thus, we can conclude that samples 1-9 have di and trinucleosomes, where as samples 10-14 have mononucleosomes and the other samples have left over DNA. Therefore, from the column trace we were able to determine that we have mononucleosomes and which samples they are in.  

Figure 2: NCP prep gel run with DNA ladder

Earlier this week I prepared samples to run through 1.2% Agarose gel. As shown in figure 2, we ran 18 samples (4-21) with two DNA ladders on each end. We ran the DNA ladders with the samples because the DNA ladders indicate the length of the nucleosomes. As you can see in figure 2, the red lines indicate how many base pairs there are in the samples, thus distinguishing whether there are  mono/di or tri nucleosomes in the samples. As we had assumed, samples 10-14 most likely have mononucleosomes. We were able to conclude this because one nucleosome has ~147 base pairs and the gel we ran shows us that those samples are closest to having the length of a mononucleosome. Even so, Dr. Andresen is going to discuss our results with Dr. Beuttner, in the chemistry department, in order to confirm what samples we should use to proceed. Even so, I believe that we will continue to purify the samples to try to get as many mononucleosomes as we can. 

Also, now that we are closer to having nucleosomes, I am working on learning how to use the Inductively coupled plasma-optical emission spectrometry machine in the lab. The ICP-OES machine is used to determine the concentration of elements in samples. Thus, I have been preparing samples to run test trials and familiarize myself with the machine as I will be using it to run actual experiments with the nucleosomes.

Week 2 - Polymer Failure

With last week's nanoparticles, Dr. Andresen and I decided to focus on citrate for now and start coating citrate AuNPs with a polymer. The reason we do this is that citrate is negatively charged, which is the same as DNA. Coating the particles with a positively charged polymer such as polyallylamine hydrochloride creates an opposite charge for the DNA to cling to when we begin to wrap it. I started this week by attempting to coat the particles with the polymer. To do this, I made a sample of polymer dissolved in a solution of 10mM NaCl so that the polymer's concentration was 10 mg/mL. I then made a ratio of 10:2:1 of citrate AuNPs, stock polymer solution, and 10mM NaCl. I didn't want to waste many particles, so I only did 10mL to 2mL to 1mL each batch. Unfortunately, during this process, I contaminated one of my original citrate batches and the whole thing turned black, rendering them useless. I still had one good batch, but I also picked up a new batch that Dr. Thompson made and used those two to make polymer coatings. Unfortunately, I failed both of my batches, with both of my samples in each batch looking horrible on the UV-Vis spectrum. What we are looking for is a slight redshift (shifted right towards the red side of the spectrum) that shows that the polymer has attached, as well as the same peak shape as the uncoated particles. The particles will obviously get a little bigger when coated with something, so if there's no redshift, the particles haven't been coated. When I finally got to the third attempt, the data looked decent. I was starting to see progress, but they still weren't great particles. At this point, I decided to try a new method from a scientific paper that someone did that was related to my topic. After trying this and collecting the results, it turns out that the new method is even worse than my original one, so I'm sticking to the old fashioned way. I also was able to make the absorption graphs in python after some tinkering with the code, and I normalized the data (made the maximum value in the data set 1 and scaled everything accordingly) in order to make the redshift clearer.

PAH-Cit AuNPs compared to their original unwrapped citrate AuNPs sample 2-1 (the red line). The blue line shows complete aggregation and is not a good set of data. As time went on, however, the method became muscle memory and the blue to grey to black lines show this progression. There is a slight redshift that occurs, which indicates growth.

PAH-Cit AuNPs in the second batch compared to the original unwrapped citrate AuNPs sample 2-2 (the black line). The same pattern is clear here as well. 

After a decently successful UV-Vis characterization, I went on to review the size DLS and zeta potential measurements. The size DLS was terrible overall, with all of my bad particles showing double peaks which indicate polydispersity (not all the particles are the same size). My good particles showed double peaks as well, however, I wasn't worried due to intensity (the y-axis on the graphs) being related to size as a factor of 10^6. This means that even though the peaks look relatively the same, the peak that shows particles with a bigger size is scaled up enormously. 

PAH-Cit AuNPs sample 2-1 3 size DLS results. The graph shows double peaking at 34 nm and 122 nm, however, almost all of the particles are around 34 nm, which is to be expected since the polymer layer only adds a few nanometers to the particle's diameter.

The zeta potential results for all of the particles were good, anywhere from 30 to 40 mV. Since they are positively charged now, it means the coating went well. However, this does not give any indication of how monodisperse the particles are, so even the bad particles had a good charge. Next week, I am finally going to start to coat my particles with DNA. I don't have very many particles left after all my tests, so I'll make some new ones to wrap. Hopefully it all goes well.

Week 2: Data Time!

Learning to use the ITC over the past week and a half has been a long process. At the beginning of the week, I got some pretty terrible results. Here's a small selection from the many failures I encountered.

Over time though, the results slowly got better. The goal is to have a smooth "hill" formed by the injection peaks which returns to a flat baseline about halfway to two-thirds of the way through the experiment. Here's a few my good data sets, which I was able to collect some good numbers from.

I was able to gather four pieces of data from this. The first is the binding energy, Kd. This is a measure of how strong the bond between two chemicals is. The next important number is the stoichiometry of the reaction, n. This represents the ratio of chemicals needed for the reaction to take place. Third, we have the change in enthalpy, delta H. This value is related to both the change in temperature and pressure/volume during the reaction. The last constant is the change in entropy, delta S. This is more or less a measure of the increase in disorder caused by the reaction.

The following graphs were obtained by plotting the area under each injection peak versus its peak number, and then fitting a curve to the data points. This gave us some very consistent results which were also pretty accurate. This is a good sign for future tests!

First week into the process of preparing nucleosomes

On Monday, Dr. Andresen and I began the process of preparing nucleosomes. We began with 50mL of chicken blood which if you’re wondering the reason behind why we use chicken blood, it’s because unlike our red blood cells, chicken red blood cells have nuclei that contain nucleosomes.

First off, Dr. Andresen and I began the process of preparing nuclei. We made 500mL of KTM buffer, which contains Tris HCL, KCl, MgCl2 and PMSF, and combined 50mL of it with the 50mL of chicken blood. Then we spinned that using a Centrifuge machine. The Centrifuge machine separates the heavier stuff (the pellet) from the lighter stuff (the supernatant) by increasing the gravitational force.Therefore, for the first couple of spins it helped us separate the red blood cells (the pellet) from the extra stuff that the blood contains such as plasma (the supernatant). Then, we resuspended the cells in KTM buffer and Triton X. The Triton X is like a “soap” which helps “break” the cells in order to release the nuclei inside of them. We had to do this because we want what's inside of nuclei, the nucleosomes.

Tuesday, we had a discussion about unproductive and productive stupidity in STEM. Right after the discussion, I actually made a mistake and added too much of one solution to a batch of KTM buffer that I needed to use in order to finalize the preparation of the nuclei. Dr. Andresen realized that I did this once he noticed how hard the pellet (which contained the nuclei) was since it wouldn't dissolve in the KTM buffer. Therefore, I had to start all over. I was frustrated at the fact that I had ruined a day of work but it actually helped me get over the fear that I had of messing up. It began my journey of understanding that research is a process of trial and error and that my mistakes will only help me learn and grow.

For the remainder of the week, Dr. Andresen and I went from having nuclei to having chromatin. When we had the nuclei, we measured how much DNA we had using the UV/VIS machine, which told us the concentration of DNA we had. We had about 125mg of DNA. Then, we resuspended the nuclei with CaCl2 + ML buffer, which contains Tris HCl, NaCl, MgCl2 and PMSF, and heated it at 37 degrees Celsius for 35 minutes. We heated this because throughout the process, the solution actually eats at the membrane of the nuclei which then breaks the nuclei and releases the DNA and everything else in the nuclei. Then, we continued by doing different steps in order to get the chromatin which consisted of making other solutions, spinning it, and letting it sit overnight with different solutions in order to get rid of the extra stuff such as ions and molecules surrounding the DNA.

As of today, Friday, we have chromatin, which consists of DNA linking nucleosomes together. Ultimately, what we’re trying to do is get the nucleosomes, which is an octamer wrapped around twice by DNA, so we'll have to cut the DNA linking the nucleosomes in order to get the nucleosomes alone. Also, today we measured how much DNA we have, which is approximately how much chromatin we have. We have 37mg of DNA which is about 30% of how much we had last time we had checked. We are hoping to at least have half of this amount by the end. However, during our group meeting today I found out that the next step is where things have gone wrong in previous years. Even so, I am hoping to have some nucleosomes next week.

Week #1: The Nano ITC

This week I've been spending a lot of time working with the Nano ITC, so I thought I'd take some time to explain a bit about how it works. After all, this is what I'll be using all summer!

Thar she blows! The Nano ITC in all its glory!

The Nano ITC, which stands for Nano Isothermal Titration Calorimeter, is a fun little box that can tell us all sorts of things about chemical reactions. In understanding what this instrument does, I find it useful to break things down word by word. First up is "Nano," which means it's pretty small. In fact, the ITC only uses about 350 microliters of sample per trial. That's about .0006 times the size of a Venti cappuccino at Starbucks! This lets us use a lot less sample to get the same information.

A view down the barrel of the ITC. At the bottom you can see the openings of the two chambers. The one in the center holds our sample and the one on the right holds our reference.

The second word in the name is "Isothermal," literally meaning "same temperature," which is exactly how the ITC works. Inside this box of wonder are two chambers, which are held at the same temperature throughout the experiment. As the temperature of our sample changes, it is checked against a reference solution placed in the other chamber, and the machine adjusts its temperature so the two match. The ITC measures the amount of power it takes to keep these two chambers at the same temperature, which gives us our results.

This is the syringe that the ITC uses to inject one of our solutions into the other.

The third word in the name is "Titration." This is a fancy chemistry word that basically means we're adding one chemical to another. The ITC uses a syringe to slowly inject small amounts of a solution into our sample. This causes a chemical reaction which we can collect data about. Also, we don't actually have to do anything after we start the machine, since the ITC does it for us! Finally, we have "Calorimeter." This just means that the ITC measures information about the thermodynamics of whatever chemical reaction is happening in the machine.
Putting all this information together, we see that the Nano ITC is a device that lets us measure thermodynamic data about a chemical reaction between two solutions. It does this by measuring the power it takes to keep our sample and a reference at the same temperature, and it doesn't even need that much solution to do it! And there you have it! That's a basic run-down of the Nano ITC. It's a really powerful tool that can give us a ton of useful information. Hope you liked the explanation, and I'll be back next week with some results from this week's testing!

First Week - Creating Nanoparticles

This week I started making gold nanoparticles (NPs) using two different methods, using citrate and CTAB surface coatings. The first is very easy to make, simply by heating up a gold solution until it boils and injecting trisodium citrate into it. This makes a red colored solution which contains the nanoparticles. The second method is much harder. The first step is preparing a "seed" solution that contains very small nanoparticles and then scaling these up in order to become bigger and easier to work with. Once the seeds have been made, they must sit for a few hours to grow and then the particles can be made by adding the seeds to a mixture of a gold solution, silver nitrate, and ascorbic acid. This is a very sensitive process, especially when adding the silver nitrate and ascorbic acid and can be messed up easily, which is what makes it difficult. However, if made correctly, the particles will be coated in CTAB and should be a red/pink color. The ones I made this week (two batches of 8 samples each) were relatively red/pink, but a few samples were purple/blue, which indicates aggregation (some of the particles have clumped together). This isn't what I want, as I want the particles to be as separate as possible in order to continue with my work wrapping DNA around each particle. If the particles are clumped, the DNA won't be long enough to wrap around and will cause problems with my experiments. I also made two batches of citrate-coated particles, which look a little redder than the CTAB ones.

Citrate-coated NPs. The middle is a nice red color, but the others are an ugly dark purple which indicates aggregation of particles.

When I ran my samples through the UV-Vis machine, which measures the absorption spectrum of the particles (how much of each color in the visible light spectrum is absorbed by the solution), most seemed decent, with a good solid peak of about 530 nanometers. This means that the particles are mostly absorbing green light / reflecting red light, which explains why the solution looks red. I had a few that absorbed way too much of other colors, so I'm not going to be using them to continue my research.

Absorption Spectrum from the first batch of CTAB-coated NPs.

I also ran my samples through a DLS machine which measures the size of the particles. Most of them were around 40 nanometers in diameter, which is a little on the large side, but it'll still work - I'll just have to correct for this when measuring how long the DNA should be. I had a few that were way too large, so I won't use these either.

The last analysis that I did is called zeta potential, and this basically measures the charge on the surface on the particles. Almost all of my particles performed well in this test and had the correct charge on their surface (about 30 millivolts). This is all well, however, the most important test is the absorption spectrum measurement, so all of the ones that did not do well in that test I will most likely be trashing. Next week, I'll start wrapping DNA around my particles and hope that all goes well - if not, I may need to create new particles.

Summer 2019 Begins with a Great New Crew

I would like to welcome Diana, Ben, and Nick, three wonderful new students to the lab. We have just begun our adventures together and already they are showing great promise. Keep checking back here for exciting developments as they tell us about their research!

Last Week

So last week Monday morning came around and Professor Thompson had been too busy to prepare gold nano-particles for me. This was fine, I was ready to tackle the task on my own. The procedure involves bringing 100mL of a solution up to a boil and then pouring 3mL of another solution into it. The solution then proceeds to change colors several times, its actually pretty neat to watch. I ended up making a pretty concentrated batch of nano-particles. It even ended looking pretty good under the UV-Vis, so I decided to continue on with my project using them. I needed to find a good ratio of NPs and PAH because the two are pretty fragile. If not mixed together properly they can immediately aggregate and make the solution unusable. Also, if they are not in the proper ratio this can also cause it to become aggregated and then unusable. So I tried to make a 3x and a 5x diluted NPs solution with normal concentrations of PAH. Both seemed usable after mixing. I then tried out the new cleaning procedure that Professor Thompson had suggested. I would spin the solutions at very high speeds, 9000 rxg and 7000 rxg in order to get all of the excess PAH out of the solution. This maximized our risk of contamination by stray PAH particles. My problem with this procedure arose when I had to wash the pellets with DNA in them. After I started this centrifugal process, I found that the solutions were creating pellets that were virtually non-existent. This is not good, as it is one of our main goals to get good pellets here. I was thinking that these pellets could not form because I had diluted the initial amount of NPs in the beginning. Professor Thompson suggested it had something to do with my spinning procedure for the DNA washes. I'm now currently trying this new procedure, which includes longer times and a larger rxg, to get better pellets in my solutions.

Continuation of Week 6

Is finally Friday! Well that doesn't matter, what matter is what I have accomplish this week. In terms of work, I have accomplish  a great deal but in terms of progress I could had accomplish more. I say this because we got our results from Monday and we didn't get the results we wanted... again. This is really frustrating but like I said last time, researching is about learning from our mistakes and improving upon them even if it takes multiple failures. Once we saw our results, professor Andresen and I came out with a new method to try out it order to see if we were doing our trial digestion correctly. Instead of using our chromatin, we instead used sonicated DNA with our trial digestion to see if we get different results in our gel. In order to do this we must first shred DNA and we would be left with a solution of liquid DNA.Then I added 50 micro-liters of sonicated DNA into two micro-centrifuge test tubes. Once this was done, I diluted the micrococcal nuclease to 100x dilution and also diluted CaC12 in order to add to our two samples. I added 0.5 micro-liters of micrococcal nuclease to one sample and to the other sample I added 2.5 in order to have two different lengths of DNA. Once this was done, I put both solutions in the heater for 15 minutes at 37 degrees Celsius and when this was done I did the same procedure as the previous trial digestions. In the end had two different samples to test in our gel, but I also made some extra samples such as DNA ladder and just the shredded DNA and the same samples with just added DI water. I added this solutions into our gel,  and when the results were ready, we again got the results which was more frustrated because it still didn't work. The results can be seen in the next picture. Figure #1

Figure #1: Results from Gel

From this picture, we can clearly see that we can't  see any base pairs from our samples, only from our DNA ladder which is located to the left. This is the problem that we have been getting on our past trials and from this picture, we think that all of our samples are staying in the beginning of gel. In other words our micrococcal nuclease is not separating our chromosomes.
After this process was done, professor andresen and I decided to do one more trial with the past solutions but this time, instead of making 3% agarose gel, we will be making 1% gel. What this would do, it would make us see the base pairs stuck at the top of gel from the previous samples. For this new trial, I will use two samples with out the proteinase K trial digestion and two other samples with the full trial digestion. Unfortunately when I was moving the agarose gel to the special the machine that was gonna give me my results, the gel felt apart and what this meant was that I needed to redo my work all over again. which I eventually did.
The next day I ran the gel and while I was waiting for the gel to be done I prepared another trial digestion that I will do next week. This new trial digestion consisted of time intervals of 5, 10, 20, 40, and 60 minutes and 1.6 micro-liters of 100x diluted micrococcal nuclease.
Once the gel was done, I look at the results and they were a little better than previous results but it still didn't show the base pairs that we want it to see. We are hoping that next week with this new trial digestion, we will be able to see the results we want to see. Its a struggle keep seeing how our trials always fail but even though its slowly, we are making progress and we will be able to find what is causing our results.

End week six

The last run of samples was supposed to be our most promising samples yet. It had looked like we were going to get not only quality data but in a large quantity as well. Unfortunately, something had gone awry. When we start with out citrate nano-particles they should be negative in charge, then we wrap them in PAH and they switch to positive charge, and then finally we wrap them in DNA and they switch charges a final time, back to negative. My first two Zeta tests were conclusive and yielded the expected results, however the final one simply doubled the number and did not switch the charge at all. Professor Andresen phoned a friend and we then scheduled a conference with Professor Thompson. Before we could have this conference however, I first had to prepare a power point presentation on my most recent bit of research. Dr. Thompson broke down several of the data pieces I am so accustomed to collecting and helped me better understand them. The UV-Vis is a lot more helpful then I know it to be, but I really need to learn how to overlay graphs on it, I am determined to learn this early next week when I have to use the machine again. The conference left me feeling very hopeful about the project. On Monday Professor Thompson is going to prepare more nanoparticles for me to use in my next batch of samples. He also suggested a much more appropriate way of preparing the PAH wrap and how to more properly purify them. I am hopeful that this will greatly improve the project. At the end of this week, I am wrapping it up by shearing DNA and looking forward to finally getting proper data next week.

Week six

So at my last post I had just found an appropriate mix of salts and nano-particles. This allowed me to then mix in my DNA and start performing equilibrium dialysis. However, I immediately ran into another big problem. What was I to re hydrate my solutions with? In the past, at this step I was not using citrate nanoparticles, but I was re hydrating with TEM buffer. I was uncertain if this would be efficient with these new particles. I decided to head to the science center and discus this problem with Rich, a fellow user of similar nano-particles. We threw a few ideas back in forth, but nothing seemed like an exceptionally good idea to re hydrate with. So I had to make a sacrifice, I gave up any hope of getting to use the ICP-OES this run in order to figure out what to re hydrate my samples with. I split my total solution of nano particles into three parts, one would be re hydrated  with mili-Q, another with TEM buffer, and a third with a solution that was 10^-2 M NaCl. Unfortunately I was only re hydrating with 4mL each when I typically do double that. Regardless, the results appeared to be conclusive, showing that the salt buffer worked the best.

So then I had to restart once again. This time, I took all of my gold nano particles and coated them with PAH. I then spun them in the centrifuge for an increasingly long period of time to siphon out the supernatant and continue spinning that. Finally, I recombined the pellets and diluted it back to 7mL with more nano particles. So I'm left with 1 super concentrated gold nano particle sample and 7 other slightly less concentrated samples. They were then all mixed with DNA and left to sit over night. Now I am currently trying to perform equilibrium dialysis, and hopefully after doing this I'll finally be able to use the ICP-OES.

End of fifth week already?

Time surely flies by when you're working full on. This week, I started working on another batch of ITC experiments after we figured out a number of flaws in our experimental protocols. Therefore, we decided to redo some of our previous experiments using different experimental parameters and protocols.
We decided that we would keep the relative concentrations of DNA and Cobalt Hexammine unaltered in the different ITC runs. The concentrations of DNA and Cobalt Hexammine used in the experiments were 2.5mM DNA+3mM Cobalt Hexammine, 5mM DNA+6mM Cobalt Hexammine, 10mM DNA+12mM Cobalt Hexammine. In addition, we decided to alter the experimental procedure a tad bit. We decided to form isotonic DNA+NaCl solutions using the dialysis method so that the concentrations of NaCl in all the samples are equal. The dialysis buffer was used for the serial dilution of the 120mM stock Cobalt Hexammine (instead of water as in the previous experiments).

After I prepared the batch of DNA samples (of different concentrations) using the regular method, I filled up dialysis tubes with 10ml of each sample. I left the tubes in a dialysis buffer of 10mM NaCl solution overnight. I used the same dialysis buffer for the serial dilution of the 120mM stock of Cobalt Hexammine in order to synthesize 10ml of 12mM,6mM and 3mM solutions respectively.

After the synthesis of the DNA samples and the Cobalt Hexammine solutions, I ran heat of dilution tests on the Cobalt Hexammine samples. Here are the results:

After running the heat of dilution experiments, I proceeded to start ITC runs with my DNA samples. I started off by using my 10mM DNA sample. Here's the overlay for the raw heat data and the integrated heat data:

That's all the highlights from week 5 folks. Stay tuned for more ITC results next week!

6th week, New trial digestion with some changes.

Wow! it's the sixth week, seems like time never stops and always keeps moving. Well today is a Monday, which are never a good feeling but we gotta hang in there. I started my day by going over the results with Professor Andresen, in order to come out with a solution or a new procedure. Eventually professor Andresen did came out with a new procedure which is the same procedure has before but with slightly different trial digestion. Instead of creating different samples of our solution by adding different amounts of micrococcal nuclease, we instead only made two  50 micro-liters of solution which contain different amounts of micrococcal nuclease. I places this solutions into the heater for 37 degrees Celsius and took out 5 micro-liters of each solution at a specific time. The different times that we assigned were 5, 20, and 40 minutes, with another sample that lasted about 2 hours. This procedure accomplished the task of creating different sizes of DNA  by letting the microccocal nuclease eat up the nucleic acids and taking small amounts of sample at different times and stopping the process by adding EDTA. Once this process was finish, I continued the trial digestion with the same procedure as before by using proteinase K and SDS. In the mean while, I created a new gel in order to put the trials in tomorrow. I also made more TBE buffer to used the next day. All this procedure took approximately the whole day due to the unfortunately broken wrist I got. All is well and I could still work, and that's really important to me because I never give up on something I start.

Final day of Week #5. Finally able to see Results

Its Friday! It is always good to come to work on a Friday because you feel at easy with yourself and work just for the fact that once this day is over, you can rest for two whole days.
Well I started my day out by making more TBE buffer, which consist of 50 ml of TBE (Tris-Borate-EDTA buffer) and 450 ml of DI water. Once I was done making this buffer I made two DNA ladder, DNA ladder is a solution of DNA molecules of different known lengths and is used to run along side our samples in order to estimate the size of our samples. Once this was done, I prepared an extra micro-centrifuge tube with only 1 micro-liter of our chromatic supernatant, 9 micro-liters of 60% sucrose and 10 micro-liters of DI water. I made this extra sample in order to see how it looks on the gel without the trial digestion. For the solutions who already had already been through the trial digestion, I took 1 micro-liter from each and added them to new micro-centrifuge test tubes with the same numbers label in order to not get mix up. I also added 9 micro-liters of 60% sucrose and 10 micro-liters of DI water to the new test tubes. Once this was done I was ready to add them into my gel. The solutions on the gel can be seen in figure #1.

Figure #1 Samples added to Gel

Once I loaded all of my samples into the gel, I set the voltage to 100 volts and waiting until a blue line reached 5 cm. While I was waiting for this process to happen, I started to unpack all of the materials that we ordered in order to have more stock available to used in the future. It was like Christmas morning, unpacking all of the boxes. See figure #2 

Figure # 2 Material needed for present and future usage

Once the unpacking was done, our shelves finally looked like they had material in it and it looks awesome. See figure #3

Figure #3 More beakers to make more Buffer

When all of this was done and my gel was finally done, I took the gel and used a special UV light to the results. Unfortunately once again we didn't get the results we wanted. After I saw my results, I felt disappointed because I had worked so hard to get better results than last time but that didn't change the outcome. Like I said before in previous blogs, being a researcher is about working hard and trying to solve problems even if it means failing multiple times. It is hard to accept the results but my results just show me that I must work harder and try again until I get the results I want. 

Week Five

The results from the last experiment I mentioned in my last blog post were inconclusive. Along with the next few experiments that had occurred after that. Now I'm not saying that it is impossible to get the data that we wanted to get with the materials that I was using, however, Professor Andresen pointed out that if the experiment was this finicky then no one would care about it enough to try to reproduce it. So later that day he found me a brand new batch of nano particles. This time we got Citrate coated gold nano particles and this changed our procedure slightly. The general idea now is to take the negatively charged gold nano particles and wrap positively charged PAH around the gold. Then we'll wrap the DNA around the PAH/Gold complex. I was very excited to get away from the old procedure that I couldn't figure out how to fix. The first problem we ran into with this new experiment was trying to coat the gold with the PAH. When I followed the old procedure something strange happened. 

Starting from the left is a 10^-2M NaCl solution, then a 10^-3M NaCl solution mixed with 10micrograms/mL of PAH, then Batch 4 of the citrate gold nanoparticles, and then on the left is a solution that mixed all of the previous three. It was very concerning that the final solution turned out purple. Something was definitely wrong, so I went to Fontaine to seek help. We tried every combination of her and my particles to see where exactly my purple solution had came from. We could not recreate the purple solution but we did eventually come out with a solution that would allow me to move onto the next step. At this point in time i'm about to mix my new Gold/PAH solution with some sheared DNA so it is looking exciting!