Author: Prof. Andresen

The rest of the week I kept washing the chicken blood with KTM and Triton X-100 in order to wash away the fat surrounding the cell. In order to wash the blood, I used 20 micro-liters of KTM and 70  micro-liters of Triton X-100 for each sample and spin it in the centrifuge with a force of 3600xg for 10 minutes at 4 degrees Celsius. Throughout the day I just kept washing the chicken blood and made more KTM because I was running out. This process kept on going from Thursday to Friday because I still had red solution in my sample which is not what I was looking for. This process continued until I can obtain white/clean nuclei. On Friday I was able to see results from washing the chicken blood and I could see white/clean nuclei for two of my four samples. I ran the same procedure for the two solutions once again and I was finally able to get white/clean nuclei for all my solutions. Figure #1 shows how the chicken blood must look after all the washes with KTM and Triton X-100.

Figure #1 White clean Nuclei

After this was done, I once again wash the clean nuclei with just KTM in order to get rid of the Triton X-100. I did this procedure twice and by the time I was done the day was over. The washing of the chicken blood takes a good amount of time due to the amount of Triton X-100 that I added but for future reference I would recommend starting with at least 80 to 100 micro-liters of Triton X-100 and go down on the volume from there.

I started out my week by once again doing the Trial Digestion chromatin due to the results we kept on getting from our DNA and Gel test. I started my work where I create samples with proteinase K, which contain 5 micro-liters of digested chromatin that I had previously created, 5 micro-liters of Proteinase K that professor Andresen recently bought, 0.5 micro-liters of SDS and for this new trial I added 0.55 micro-liters of CaC12 to the sample. After all this solutions where added, I let the samples sit for 50 minutes at a temperature of 50 degrees Celsius. While I was waiting for this to be done, I started preparing the gel once again. Once the gel was done, I placed the gel on the refrigerator for about 1 hour and 30 minutes. Figure one depicts the machine use to heat up the samples.

Figure #1 Isotemperature

Once the 50 minutes were up, I took out the samples and placed them into ice. By this time I got new micro-centrifuge tubes and took 1 micro-liter of solutions from samples and added it to the new test tubes. I also added 9 micro-liters of 60% sucrose and 10 micro-liters of DI water. I did this for each sample that we had, and I also added 1 micro-liter of blue dye in order to see it once we added into the gel. During this time I also made DNA latter which contain about 4 micro-liter of DNA ladder and 10 micro-liter of 60% sucrose. During this time I took out the gel out of the refrigerator but notice that I used the wrong Comb! and this was really bad because due to this I needed to redo the gel and this took about the whole day. Once I was done making the gel I let it sit on the refrigerator though out the night. In my spare time I kept washing the new chicken blood with KTM. Figure # 2 displays the right comb to used for the gel.

Figure # 2 Comb used to make holes in the gel.

The next day I came back, I took out the gel out of the refrigerator once again and this time I had everything  ready to go so I put my samples in the gel and used a machine to provide voltage in order to move the DNA from one side to another. This process took about 5 hours and in the mean time I kept washing the Chicken blood with KTM. Once I came back from lunch, my gel was ready to be view by a UV machine. My results showed a better improvement on the last trials but It still wasn’t good enough for what we were looking for. Now professor Andresen and I decided to redo everything from the beginning but this time we will used the new fresh blood that was bought recently  and see how this results come out and compare them our previous results. We aren’t so sure why our results came out the way they came out but we are thinking it might have been with storing our chicken blood in the -80 degrees Celsius refrigerator might have something to do with it. I guess this is part of being a researcher, to keep trying and improving on previous results in order to progress and grow both has a researcher but also has a human being.

Since my last blog post I have not been able to move forward from the equilibrium dialysis step. I have restarted back to square one twice since then. However, we are continuing to struggle with this step. On the last run we got one of the four samples to actually provide sufficient data so currently I am trying to run eight samples in order to get more sufficient data. I’ve also been heavily experimenting with running the centrifuge at different speeds to see if that could help optimize the data. I am also eternally grateful for professor Andresen setting up his centrifuge machine in our lab. This saves me from sprinting back and forth from the science center and Masters Hall a hundred times a day. When we switched machines however, I had to figure out the RCF (relative centrifugal force). Where two machines at the same rpm can provide varying forces on the things inside, RCF is constant between everything. So after switching machines I upped the RCF by about 1.5x per run, in order to see if this would yield better data. I could then run the supernatant or sample in  UV-Vis machine to see where both the gold and DNA was and had gone too. This allowed us to conclude that spinning at 3900 RCF was the best option for us right now. With my current eight samples that I’m using, I initially spun all eight of them, but spun four of them an additional time before taking the supernatant off and re hydrating. When we finally get data this should allow us to see which method works better. I have another spin or two to finish by the end of today but I am hoping to start Monday off with getting some great data!

I started my week by getting my hands in chicken blood that I was going to used to extract its DNA and prepare mononucleosomes. The amount of chicken blood that I got from a refrigerator at -80 degrees Celsius  was about 50 mL of blood solution and 25 mL of blood cell volume. Then I added KTM with PMSF (Used to keep proteins from destroying the cell)  already added in the KTM to have a total of 100 mL of blood and KTM, after this was done I split the solution inot 4 x 25 mL solutions. This can be seen in figure #1.

Figure #1 Chicken blood and KTM

After these solutions where made, I had to measure their weight and make sure all of them weight the same mass, this needs to be done in order to used the Centrifuge machine. The centrifuge machine works by spinning solutions at high speeds which makes the gravity increased around the solution and helps separate liquids that have different weights. For our solutions, this process separates blood cells from plasma cells. Since we separated our solution into four different blood samples we only needed to get he same weight for each of them and placed two solutions on each side in order for the solutions to be balanced in the Centrifuge. If for some case we only have one solution, we need to used water has our other sample in order to used the Centrifuge and have a balance sample. Once we spin our sample in the Centrifuge at 3900xg for 5 minutes and then removed the supernatant carefully. The supernatant is the liquid that is on top of the nuclei which is shown in figure #2.

Figure # 2 Supernatant after spin

I did this procedure four times, and after this was done I added 0.2% of Triton x-100, which is a substance used to wash away the fat surrounding the cell. Then we spin the solutions in the Centrifuge at a speed of 3600xg for 10 minutes at a temperature of 4 degrees Celsius. When this was done we incubate for 30 minutes on ice while turning the tubes every 5 minutes. This process took the whole day, and so after all of this was done we placed it in the refrigerator and came back to it the next day.

The next day came and I did the same process has the previous day, I measure the mass of the four solutions we had and added Triton x-100 to wash our solution once again. I repeated this same procedure until I obtain white/clean nuclei. This can be seen in figure #3.

Figure #3 White/Clean nuclei

 One I got this results, I added the four solutions into two test tubes and removed all supernatant. On this two test tubes I added KTM which includes PMSF in order to wash and get rid of the Triton x-100, and I did this twice. When using the Centrifuge, I set it to 3600xg for 10 minutes each time. When the washing was done, I added 8mL of ML and spin it at 3000xg for 5 minutes at a temperature of 4 degrees Celsius. I did this process four times and each time I measured the mass of each test tube in order to use the Centrifuge. This process once again took the whole day, so after I was done with this, I placed the solutions into the refrigerator.

The next day I took 2 small samples of the solution and used the UV-VIS in order to determined the DNA concentration, we used the wavelength of 260 and 320 and determined its absorbent.  In the beginning I was having trouble with the UV-VIS because of issues with the computer software, the problem was solve by restarting the computer once again. This procedure took about two days just because myself and Professor Andresen weren’t sure if the data was correct. Once again we did this procedure by taking 10uL of nuclei from our solutions and then adding 930uL of H2O and then adding 50uL of 2M NaOH and 10uL of SDS. Our results can be seen in figure #4.

Figure #4

Once we got this results, I re-suspended the nuclei in 15mL of ML and PMSF, and split it into 7mL in each 15 mL tube. This solutions were then spun at 3000xg for five minutes at 4 degrees celcius and discarded the supernatant once again. This was done three more times.  When this was done I needed to bring our solution to a temperature of 37 degrees Celsius. Since our nuclei was about 18 to 19 mL, I needed to split it into 4.5 15mL tubes in order to fit it into the Iso-temperature machine, and this was left in the machine for 10 minutes. Once the 10 minutes were off, professor Andresen added 8.3uL of Micrococcal nuclease and we let it seat in the machine for 30 minutes. After the 30 minutes, I added 368uL of EDTA in order to stop the reaction of cutting the DNA. I ice it for 10 minutes and then pour the solution into one test tube, proceeded by placing it into the centrifuged for 5 minutes at a speed of 1000xg at 4 degrees Celsius. I took out the solution from the centrifuge once it was done and I removed the supernatant and kept it. I also made 500mL of EDTA with a concentration of .250uL of EDTA and the rest was water. The next part was done by Professor Andresen because it takes a lot of skills and precision to get it right on the first try. He used Dialysis clip and Dialysis tubing to create a sort of bad for the nuclei to rest at. The dialysis bag is used to let liquid through but not anything else, once this bags were created, they were placed into the 500mL of EDTA and left overnight on the refrigerator.
Friday I started my day by getting training in laboratory safety procedures and then once this was done I headed to the lab. Once I got into the lab I took out our solutions from the refrigerator and from the EDTA that was placed in and pour it into two tubes. I took out a small sample from each solution to see how much DNA concentration we had and we did this by using the UV-VIS has previously done. Once the concentration was found we put our solutions into a stronger centrifuge in order to spin it to a speed of 8000xg for 20 minutes in order to spin down the foggy membranes and debris. When this was done, we should had 80-90% of the total post-digestion DNA in the supernatant and the other percentage in the pellet. I combined the two supernatant from both solutions into one tube and added concentrated stock to make 50mM of NaCl. We placed our solution into a beaker with a spinner and left it stir it slowly at 4 degrees Celsius for the weekend. We will check back at our results in the next week.

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.