8. 8 Sample GMM (Red)Materials were kept on ice during the PCR preparation. We added 20 µl of the indicated master mix to each PCR tube, cap tubes. Later 20 µl of the indicated DNA was added to each PCR tube. Avoid InstaGene® pellet at the bottom of the tubes. Place PCR tubes in thermal cycler.<br />This thermal PCR cycler exposed the samples to three steps: denaturing, annealing and elongation. In the step of denaturing the samples were exposed to a temperature of 94˚C in order to separate the two strands of the DNA. Then the temperature cool down to 59˚C and at this moment is when the primer began to stick to the specific places of the DNA in order to initiate the replication. Next the temperature raised to 74˚C and at this temperature was when the Taq polymerase began to stick the nitrogenous. It was done 40 cycles after the initial denaturation but before the final extension. Approximately this part of the experiments lasted 4 hours. <br />Electrophoresis <br />For gel electrophoresis, a 3% Agarose gel was used. This gel was made by first mixing 7.5g of Agarose powder to 250mL of 1% TAE buffer. This solution was mixed until the Agarose dissolved completely. The solution was then heated in microwave oven in intervals of 1 minute, where the solution is heated then stirred. This procedure was repeated until bubbles start to from the Erlenmeyer flask. Once the bubbles form, the solution was covered and left to cool. Note: the solution prepared had some undissolved clumps and was stirred to dissolve it. After the clump was dissolved, the solution was poured unto a casting tray. With a clean pipette tip, bubbles that could affect the results were moved away from the center to the sides. The casting tray was wrapped in plastic and then placed in the refrigerator for one day. <br />The next day involved the insertion into the wells of the gel. First, the gel casting tray is assembled unto the electrophoresis chamber. The eight DNA samples that were amplified were added 10µL of Orange Loading dye. The Loading dye gives the samples a little more weight, so the samples don’t float away. Before dispensing the samples to the gel, 1% TAE buffer was deposited unto the electrophoresis chamber until the point where the buffer was level to the gel. For the electrophoresis, not only did we put 8 DNA samples, but also 2 rulers to measure the bands. Using an eppendorf micropipette, 20µL of each sample was inserted into their corresponding well in the gel. After having depositing each of the samples, the electrophoresis chamber closed and the electrodes are connected. The gel ran for 30 minutes on a constant voltage of 100V. Gel ran for 30 minutes at 100 V. Afterwards it was stained with Ethidium Bromide and visualized using UV light.<br />Results<br />The results of this research can be simplified in an electrophoresis gel. <br />Figure 1: This gel is for Triticum spp. and Zea mays. We were supposed to see the bands of a ladder focusing on 455bp for plant molecular marker and 203 bp for the GMOs.<br />Figure 2: This gel is for Carica papaya and Glycine max. We were supposed to see the bands of a ladder focusing on 455bp for plant molecular marker and 203 bp for the GMOs.<br />Discussion <br />We identify obvious degradation of DNA material. For this reason the entire experiment didn’t prove or revoked our hypothesis. Human errors are involved and affected the results of the investigation. Pipetting, concentrated DNA, lots of primer, evaporation, and contamination are among the possible errors that occurred during this experiment.<br />References<br />Cantamutto, M., & Poverene, M. (2007). Genetically modified sunflower release: Opportunities and risks. Fields Crops Research, 101, 133-134.<br />Jiao, Z., Deng, J., Li, G., & Cai, Z. (2010). Study on the compositional differences between transgenic and non-transgenic papaya(Carica papaya L.). Journal of Food Composition and Analysis, 23, 640-647.<br />Scipioni, A., Saccarola, G., Arena, F., & Alberto, S. (2008). Strategies to assure the abscence of GMO in food products application process in a confectionery firm. Food Control, 16, 569-578.<br />Terzi, V., Malnati, M., Barbanera, M., Stanca, A., & Faccioli, P. (2003). Development of analytical systems based on real-time PCR for Trititcum species- specific detection and quatitation of bread wheat contamination in semolina and pasta. Journal of Cereal Science, 38, 87-94.<br />BioRad® Manual of: Biotechnology Explorer™ GMO Investigator™ Kit<br />