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DNA, also known as deoxyribonucleic acid, it is the genetic make-up of mostly every organism. This genetic information in DNA is found in a certain genetic code that is made out of 4 bases adenine, guanine, cytosine, and thymine. These bases, couple up with each other making base-pairs, cytosine with guanine and adenine and thymine. The way these base pairs and are sequenced is what determines the genetic make-up of each organism and how they will develop, making them unique. DNA has a sugar-phosphate back-bone, this with the base pairs form the double helix structure of DNA. Most DNA is found in the nucleus of most cells and some in the mitochondria of the cell.
The DNA that is within the cell nucleus it is in the form of chromosomes. These chromosomes are just pairs of tightly packed chromatin pairs. Chromatin are strands of genetic information and are made-up of DNA and histone proteins, this proteins help in the process of condensing the large volume of DNA into a smaller volume that its able to fit the cell nucleus. DNA exists as chromatin because as the chromatin condenses and packages the DNA inside the cell's nucleus, it protects the DNA during the processes of either mitosis or meiosis. It is found inside the nucleus of eukaryotes. In this experiment we are extracting chromatin from both plant and animal cells, which are both eukaryotes.
This experiment was done in two parts, the first part concluded of the extraction of chromatin from plant cells and the second part was the extraction of chromatin out of animal cells. Frozen strawberries were used for the first part of the experiment and calf liver was use for the second part. The processes of extraction of chromatin from both plant and animal cells were very different between the plant and animal cell. Although both cells are eukaryotes and each have a nucleus that contains chromatin within them, there are some physical differences that may affect how different both the procedures of the extraction of DNA are. A main difference between the cells is that plant cells have cell walls, which is something animal cells do not have, this may cause for some physical force to be put on the strawberries to break down the cell wall. This is something that will not be needed to do for the animal cells. Another difference between the cells is that plants such as strawberries tend to be polyploidy, this means that they have more copies of their genetic information per cell when compared to animal cell such as the cells within the calf liver, this may affect how much chromatin we are able to extract from plant and animal cells.
There are also some chemical properties within DNA and chromatin that will have an effect on the procedure, such as the negatively charged sugar-phosphate back bone of DNA allows it to be able to dissolve in polar solvents such as water. That is why in this experiment by adding salt to the DNA solution the anions of salt are able to disturb the negative charge of DNA and allow it to less be soluble in polar solvents. As the solvent we used ethanol because it a less polar solvent and it allows for the chromatin to precipitate easier and allows it to be separated from the solution. The purpose of this experiment successfully extract chromatin from each type of cell and then analyze and compare the procedures and result of each part of the experiment.
The first part of the experiment consisted of the extraction of chromatin from the frozen strawberries. We used frozen strawberries because strawberries are octoploid meaning they have sets of 8 chromosomes in each cell, which is more than other cells. They were frozen because ice helps disrupts the cell. We mashed up the frozen strawberries to physically break the cell wall. As mentioned previously DNA it is negatively charged and because of this it becomes soluble in polar solvent such as water. That is why our solution concluded of warm salted water and detergent, the salt was added so that the anions would shield the negative charged of DNA and make it less soluble in the solution and allow precipitation. The detergent was use because it helped disrupt the cell membrane after the cell wall had been destroyed and help inhibit DNA degrading enzymes (DNase) released after the cell had lyzed. This helped DNA to be released. After the solution is filtrated the filtrate mainly contains chromatin, salt water/ detergent solution, and small protein particles that were able to go through the filter. The left over pulp would mainly consist of proteins. We then slowly added ethanol to the filtrate because we wanted to form two layers; ethanol is a less polar substance which allows the chromatin to precipitate better. Once the chromatin precipitated it appeared to be a solid, clear-white-ish, spongy, string-like substance floating in the ethanol, after the resulted substance was removed from the ethanol it was heated for a few minutes. After the heating the ethanol evaporated and the chromatin shrunk so much that it was very hard to see, this could be the result of the chromatin breaking down.
The second part of the experiment was the extraction of chromatin from the calf liver, the preparation of the calf liver mixture had already been done for us by our TA prior to lab. This calf liver solution contained 250mL of EDTA-saline and 50g of calf liver. The EDTA chelates metal ions, making them not able to perform as cofactor for DNase, DNA degrading enzymes, by in a way inhibiting the DNase, the DNA will not be destroyed after the cell has lysed. After the mixture had been blended and allowed to sit for a few minutes it had to be filtered to separate as much cell debris from the solution. The solution was then put in a centrifuge at 2,000rpm for fifteen minutes. After it was centrifuged, the denser material of the mixture was at the bottom of the tube, also known as the pellet, and the less dense material made-up the liquid solution, called the supernatant. The supernatant that was formed was disposable because at that point the pellet was what contained the chromatin and some cell debris. Once the supernatant was measured and discarded, that same volume of the supernatant that was discarded was then replaced with EDTA-saline. It is once again centrifuged, supernatant is once again discarded. This had then concentrated our pellet to mainly chromatin and some debris. The pellet was then mixed with 40mL of 2.6M NaCl, which was used to shield the charges of the DNA, so that it will be easier to extract the chromatin. The concentration of NaCl was important because we needed a trong enough concentration that would shield the negative charges but low enough that it wouldn't destroy the DNA. The mixture is then placed in a machine that suspended the solution it at a high speed to dissolve the chromatin. It is then placed on the centrifuged for the last time, this would then result in the supernatant containing the dissolved chromatin and the left over pellet consisting of any leftover cell debris and possibly DNA that wasn't able to be dissolved. Because the supernatant finally holds dissolved chromatin, it is then separated from the pellet and set aside (not being discarded).
Cold 95% ethanol was then slowly added to this mixture, the purpose of slowly adding the ethanol was not to disturb the layers. It was important that we added on a certain amount of ethanol that would dilute the concentration of the solution to be 70% ethanol. The purpose of this was because this concentration of ethanol (70%) is high enough to be able to precipitate the mainly DNA and not a considerable amount of proteins. To find the volume of 95% percent ethanol that was needed to be diluted so that the final concentration of ethanol would be 70%, we used this formula: C1V1=C2V2. After using the correct volume of 95% ethanol that came up to be about 112mL of 95% ethanol, we were able to see a precipitate form. When comparing this precipitate to the strawberry precipitate there was a clear difference. For the strawberries we were able to see a more clear structure of the chromatin and actually feel it. For the calf liver we only saw a white-ish precipitate form between two layers of the solution but the structure wasn't really visible. This could have been the result of the genome available in each cell or just simply because of the way the procedure was carried.
In conclusion, it was clear why both these procedure were so different not only because of physical properties but also chemical properties that cause for extra or different steps during the procedures.