Tuesday, June 13, 2017

5th week, Continuation of the preparation of Mononucleosomes

I started this new week by re-suspending  my nuclei in 8ml of ML and spind it down at 3000xg for 5 minutes. I did this process 4 times in total and all of this is done for the preparation of Mononucleosomes. Once this was done four times, I removed the supernatant and took a small sample in order to check for how much DNA concentration I had and added 2M of CaC12 to make 1mm. Once this was done I need to make a calculation of how much Micrococcal nuclease to add to each sample. Micrococcal nuclease is used to eat up the DNA strands until it reaches  the nucleus. Once I found the correct amount of Micrococcal nuclease to add, I pre-equilibrated  the solution to 37 degrees Celsius for short DNA fragments for 30 minutes. Once the timer is up, in order to stop the reaction, I must add 10mm of EDTA, for my specific amount of solution, I added 161.488 micro-liters of EDTA. When all of this process was done, I was once spun the solutions at 1000xg for 5 minutes at 4 degrees Celsius. The final I step I needed to do was to create dialysis bags and let them sit over night, In my case I made 4 different dialysis bags, 3 containing our supernatant and the other containing the nuclei (left over white solution). The next pictures show the tools used to make the dialysis bags.
Dialysis  tubing and Clips.

Dialysis bag 
Dialysis bags, all inside 500mL of EDTA. This must be left over night.
Dialysis tubing and clips are used for the separation of small molecules from macro-molecules.
The next day I came back I took the dialysis bags out from the EDTA and put the 3 liquid samples into test tubes and the white nuclei I also put it in a test tube. The next thing I did was to spin each solution in the centrifuge for 8,000xg for 20 minutes. Once this was done I took out the supernatant and calculated its DNA concentration for each. The next thing I did was to combine both my supernatants together and then calculated how much DNA concentration was in this new product. When this was done, I added 1875 micro-liters of NaCl, 1800 micro-liters of EDTA and .9 grams of CM Sephanex. Once these components were added, I let the solution stir slowly at 4 degrees Celsius for the night. I felt really proud of myself because I was able to due all this solutions by myself. In the previous preparations of these same solutions and procedures , I was learning from the great skills that Professor Andresen had demonstrated and now I am capable of doing such great task. I have learned so much this previous weeks and I am still learning a lot from my mistakes and results. 

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