Sunday, May 28, 2017

Week #2 Preparation of Mononucleosomes

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.

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