The materials used included those involving a) Lab equipment: Gel electrophoresis chamber (Mini-Sub® cell GT systems, Bio-Rad Laboratories, Inc.), PowerPac™ Mini power supply (Bio-Rad Laboratories, Inc.), Thermocycler (PTC-100 MJ Research, Inc.), MJ Mini Thermocycler (Bio-Rad Laboratories, Inc.), Fisher Vortex Geniez TM (Fisher Scientific, Inc.), Thermo Scientific Sorvall Legend Micro21 centrifuge, Bio-Rad Minicentrifuge (Bio-Rad Laboratories, Inc.), Bio-Rad Professional Adjustable (Volume Micropipet, 2–20 µl, 20-200 µl, 100-1000 µl. Bio-Rad Laboratories, Inc.), and Gel Imaging System (BIO RAD Gel Doc EZ Imager).
The second set of materials were, b) Reagents: Syber safe gel stain (Invitrogen by Thermo Fisher Scientific), Ladders 9information was obtained during lab), and Ready-to-go™ PCR beads (Fisher Scientific).
The methodology used entailed, sequentially: a) each group was given a plant type. The plant type for my group was Wild-type and therefore a leave was extracted from the Wild-type plant. A tissue of close to 0.5 inches in diameter was then removed, b) then the extracted sample was then placed in a micro-centrifuge tube with an appropriate label D3-W whereby D3 was my group code and W the chosen genotype, c) and about 100 μL of nuclei lysis solution was titrated into the micro-centrifuge. The purpose of this solution was to break up the organelles that are bound to the membrane of the cells, d) then a plastic pestle was utilized in mashing the tissue inside the tube for close to 2 minutes. This assisted the nuclei lysis solution to effectively penetrate every cell in the extracted tissue, e) about 500 μL of nuclei lysis was again added to the tube thereby bringing the total volume of the solution to 600 μL, f) the tune was then incubated in a clear water bath at close to 65ºC in separate room for close to 15 minutes.
This process of heating helped in catalyzing the destruction of organelles bound to the membrane by action of nuclei lysis solution, g) about 3 μL of RNAse solution was then added to the mixture in the tube and the resulting solution mixed. The addition of the RNAse solution helped in the degradation of the RNA that would in the process have led to serious interference with the process of Polymerase Chain Reaction, h) the sample was then incubated in another water bath having a temperature of 37ºC for strictly 15 minutes. The heating process in this stage aided the previously added RNAse solution in breaking down the RNA though the catalysis of the chemical reaction between RNAS and the enzyme RNAse, i) the test tubes were then left on a rack for cooling to take place after the process of water bath, j) the next process involved the addition of close to 200 μL of protein precipitation solution. The tube was then vortexed for close to 7 seconds so as to achieve a thoroughly mixed solution. Then the protein precipitate solution was utilized in separating the protein precipitate from the tube’s liquid content.
k) After vortexing, the tubes were then put on ice for close to 8 minutes so as to cool them to the required centrifugation temperature, l) after close to 5 minutes on ice, the tube was then positioned in a balanced centrifuge, m) the centrifuge was then activated and spun for 4 minutes so as to achieve a separation of the preferred FWA gene from the protein and cell debris. After extracting the tube from the centrifuge, the debris was clearly visible as distinct precipitate pellets at the tube’s bottom.
The supernatant contained the DNA, n) about 600 μL of the required supernatant were then moved into a clean tube of 1.5mL capacity and appropriately labeled as “D3-W”. This process was important since it helped in the separation of DNA from all cell debris. Additionally, it was important to extract the supernatant meticulously without disturbing the pellet at the tube’s bottom, o) a 600 μL solution of pure isopropanol was then added to the new tube, p) the tube was yet again placed in a centrifuge and spun for a full minute. A pale pellet layer was then visualized at the bottom of the tube and it comprised of the DNA. The supernatant in this case was the unwanted protein and chemicals, and lastly, q) a micropipette was then used to extract the supernatant in various measurements. The process was done carefully in order to avoid pipetting the delicate pellet. A waste tube was used to expel the supernatant.