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So like I last mentioned, I did start off Monday by characterizing the DNA in Professor Thompson’s UV-Vis machine. The peak wavelength was at 527nm, this is a good sign. A solution with this peak wavelength indicates that the majority of the gold nano-particles are the size and shape that we want them to be. I then used a quartz cuvette in the UV-Vis machine to examine the DNA. The peak was then at 258nm which is a good place for the DNA’s peak to be. We also recorded the wavelengths at 260nm and 320nm. This was done because subtracting the 320 wavelength from the 260 wavelength can allow us to calculate the concentration of the DNA in the solution.

Next I began preparing for equilibrium dialysis. First I prepared some TEM buffer, which is like TE buffer but slightly different. It contains NaCl, Tris, EDTA, and water. This would be what is used to re-hydrate the solutions during dialysis. Then I did more calculations to find the proper ratio to mix the DNA and gold nano-particles together. Unfortunately (?) something must have gone awry, for the DNA and gold nano-particle solutions (which all SHOULD have contained exactly the same thing) looked very different.

It’s very clear that these solutions are not all the same. We decided to re-run these samples in the UV-Vis to try to identify where things had gone wrong. The UV-Vis showed us that the samples were all relatively the same, so we continued to equilibrium dialysis. For equilibrium dialysis we use big centrifuges located in Professor Thompson’s lab. The procedure calls for 40 minutes at 3000rpm. Unfortunately, I ran the first 40 minutes at 3000rpx (?) which is roughly 5000rpm. This is much more forcible then what should be used, and likely caused the DNA to crash out. After the first spin it looked like this:

To me, this looked pretty normal. The pellet is not very big, but is definitely noticeable. The supernatant was then siphoned off and the solution was rehydrated with TEM buffer. The second run, I was sure to set it at 3000rpm for 40 minutes. After the second run the solution looked like this:

It is very noticeable how small the pellets are and this concerned me. I then siphoned off the supernatant and re-hydrated the solution with TEM buffer and ran the solution at 3000rpm for 40 minutes in the centrifuge one last time. After this last run, the pellet was almost unnoticeable. Regardless, I siphoned off the supernatant and re-hydrated the solution. Because the pellets were so small on the second and third runs, we figured that something was wrong. To try to find what had gone wrong, we decided to run everything in the UV-Vis machine again. After getting the results back, it appears that the first run at 3000rxm had in fact caused the DNA to crash out.

Introduction to Lab requirements and goal

The first week working with Dr. Andresen was a great success and great experience. We started our Research with a basic understanding of what our goal was and proper ways to work in an environment where all data must be collected and solutions must be carefully made.

Monday, May 15, 2017

On Monday I met with Professor Andresen and his summer research group to discussed the plan and the goal for the research that we are investigating. We went over the basic requirements that we need to accomplish such getting keys to lab room so we can have access when Professor Andresen can’t make it. We also went over the websites that our group must used in order to keep track of our progress and find preparation guides for solutions that we might need in the near future. This website is called lab wiki and we can find useful information on how to specifically do a solution and it also gives us the freedom to create our own page where we can post a guide on the techniques that we used to create a specific solution. I was given Abby’s and Sarah’s work book to gather information on their procedures and work done previous summer’s. I was also given articles and books related to the cell and specially Nucleosome Core Particle to learn more about how the cell works and the role of DNA.

Tuesday, May 16, 2017

On Tuesday I made 50ml of 1 Mole stock of Na-Cl which is about 2.922g. After creating this solution I added the Na-Cl into a disposable container  of 50mL and label it with the name of the solution, my initials and the day it was created. This was also done to 1 M of Magnesium Chloride hexahdrate and I used 10.165g of stock  creating 50ml. After creating this solution I went to the wiki page and look for the preparation of Tris-HCl. Printed our the instructions and taped them into my workbook, and I made the solution following each step with the help of Professor Andresen, since I was dealing with powerful acid that I wasn’t used to work with. This was done through out the day, seeing how I was learning how to used the proper techniques and tools to complete each task, this was all done with the help and guidance of Professor Andresen.

Wednesday, May 17, 2017

I started out my day by creating 50ml of EDTA with about 1/2 molar, this translate to 9.3004g of stock. On this day Professor Andresen added NaOH to get the content to an pH of 8.0. While the Professor was doing this, I was left the task to make 500ml of KTM, Ml, and DB solutions. Each solution contain different components such has Tris-HCl, NaCl, and MgCl2. The procedure in making this solutions can be found in documents provided by previous research members. This solutions and data took the entire day, careful measurements were made and tools used were washed to keep a clean laboratory and environment.

Thursday, May 18, 2017

I was left with the task to make more Tris-HCl with pH of 7.5. From what I learned from Professor Andresen, I took careful measurements of the amount of HCl I added to the Tris-HCl in order to control the pH to be 7.5. I also was very careful in handling HCl, by wearing protective gloves and plastic glasses. After creating 50ml of Tris-HCl, I added the procedure to make EDTA onto wikipedia. After this was done I helped Dylan when using the UV-VIS machine, but we encounter some software problems that created problems in our solutions for the nano-particles. The time I had between projects, I read over all the documents that I still had from Professor Andresen, and read more about the structure of DNA in a cell.

Friday, May 19, 2017 

On this day we started by having a group meeting with Professor Andresen and my colleagues. This group meeting was to inform Professor Andresen about our progress in our work and an overview of what we had done during the week. On this day we also got information on software that will be helpful in future research of scientific articles and citations. When this was one, my colleagues and I went to the Science Center to shred DNA and we took careful notes on every step we did. When this was done we launch the DLS machine and got results from the nano-particles with used. During this time we also got more nano-particle from last summer that Savana was using, and to finish the day off I spend relocating the nano-particles into new containers and reading useful articles that Professor Andresen provided, in order to prepare for next week.

We picked up this summer almost exactly where I left off my training with Savannah. Since I first started, the organization of the lab had been bothering me. Especially the bookshelf, I just did found the entropy to be too high. This is no longer the case! A very thorough cleaning has left us with several empty drawers, an alphabetized bookcase and an alphabetized cabinet of  samples. Additionally, I prepared all of the samples required to calibrate the ICP and the calculations required to do so. This process included massing several solutions and then diluting them to a specific concentrations. After I had done this Professor Andresen believed that I was qualified enough to write my very first Wiki article on the subject. The process of creating my very first wiki page was exhilarating, it feels almost like a child to me now. Today, Wednesday, I was almost able to set up the ICP machine by myself. My goal for today is to run the ICP on the samples that I prepared yesterday. Below is a picture from my notebook of the theoretically calculated masses of the samples I prepared and below the theoretical masses are the actual ones.

This week was my final week of research this summer. I was very busy all week trying to get as much done as I could before I had to leave. On Monday I reran the DLS for the new cleaned DNANP samples and saw the same dramatic increase in size following cleaning. We still do not know why this phenomenon is occurring in the new samples but was not seen the original (10 ug/mL) sample. There are not any obvious signs of too much aggregation in the salt-TE cleaned samples so it is unclear why the phenomenon is occurring at all.

                                  

I didn’t have ICP gas to run my samples from last week spin cleaning so instead on Monday I ran UV vis on the supernatant samples to confirm that the free DNA concentration decreased with each spin. The free DNA in solution plummeted after the first spin so I am confident that the spinning protocol is enough to clean the free DNA out of the solution leaving only bound DNA- NP complexes.

                       

Today is my last day in lab for the summer. In the beginning of June, I began by ordering a stock of whole chicken blood, and proceeded to purify this stock throughout the summer. For the past few weeks in particular, I had been working on running gels to digest the sample with the correct amount of micrococcal nuclease. This week, I correctly digested both of my samples, so they are now ready to be run in a column to isolate the mononucleosomes. In the fall, I look forward to further purifying my samples until I just have mononucleosomes, and running electrostatic experiments on them!

For the past several weeks, I have been working on “digesting” my sample, and running sample os varying degrees of digestion with gel electrophoresis. The first step of the process is to do a “trial digest,” in which different concentrations of micrococcal nuclease are added to the nucleosomes. Micrococcal nuclease effectively “eats” the DNA, slowing down when it approaches the histone core. A higher concentration of micrococcal nuclease will “eat” more DNA, so the optimal amount that will digest only the linker DNA is sought.

Next, proteinase K is added to the nucleosomes, which digests the histone proteins. There is now free DNA in the sample, and its length is determined by the amount digested by the micrococcal nuclease. Gel electrophoresis can be done on the variously digested samples to qualitatively see how long the DNA is for each micrococcal nuclease concentration (proteinase K concentration stays the same). The goal of this “trial digest” procedure is to determine what concentration of micrococcal nuclease digests just the linker DNA, so that all we are left with in the sample is the histone core and DNA wrapped around it. It is known that there is approximately 146 bp of DNA around a histone core (without the linker DNA).

It took several attempts to get a successful gel though. Examples of successful and unsuccessful gels are below.

This gel is an example of a gel that was not successful. It is a good gel in regards to the quality of the 10 bp DNA ladders (in the first and last lanes), but the samples are all trapped in the wells, rather than moving down the gel like the DNA ladder did. Such behavior could be explained by improper digestion, perhaps because of the micrococcal nuclease itself, its digestive medium, or other factors.

This gel is an example of a successful gel! Although the DNA ladder is not as clear as the gel above, the samples did not stay in the wells, and instead, moved down the gel depending on their length. It is interesting, though, that a majority of the digested samples have a length of approximately 300 bp (double what it should be!).

Because the 40 units of micrococcal nuclease seemed like the optimal concentration to digest the DNA, the sample was digested with this concentration, and compared in another gel to the prior undigested sample (see image below).

This gel contains the DNA ladder (not very clear), the digested sample, and the undigested sample. It is good that there is a stark difference between the digested and undigested sample…but where exactly is the undigested sample? Is there DNA there, is it over digested, or does the concentration of DNA in the gel sample need to be increased?

The goal this week was to clean all of the unbound DNA out of a stable DNA-nanoparticle solution so that we can quantify the amount of DNA per nanoparticle. Due to the results we saw last week we used 10 ug/mL DNA in the same 0.3 nM CTAB gold nanoparticles. The protocol we came up with was to spin down the nanoparticles and then do 3 washes with TE+10mM NaCl followed by 2 washes with pure water. While doing this I encountered some aggregation and incomplete pelleting. The aggregation was reduced after each spin indicating that the spinning was clearing out any unstable particles from the solution. The incomplete pelleting was combated by spinning the collected supernatant 1-2 extra times and collecting the extra pellet and a hopefully clean supernatant. The result of this protocol modification was 3 times as many spins. Each spin took 40 minutes so the protocol took two very long days to complete. The result was two DNA-nanoparticle samples a sample washed by salt-TE buffer and water and a sample washed only with salt-TE. More aggregation was seen after adding cleaning with the water indicating that either the salt or the TE buffer is integral in maintaining the stability of the nanoparticle complexes.

Failed gel