Materials and Methods
To begin this experiment, we first chose which food we would be testing, which was watermelon in our experiment. We then ground one gram of our control food, which was oats in a mortar and mixed it with five milliliters of distilled water to form a slurry and continued to stir and grind the mixture. Once the oats were mixed in with the water, we added another twenty five milliliters of distilled water to dilute the mixture. Using a transfer pipette, we put fifty microliters into a test tube already containing five hundred microliters of InstaGene Matrix and labeled the tube as “N”. We capped the tube and vortexed it to ensure that all of the liquid was mixed. We then washed and dried the mortar to prepare for our next test.We then weighed out one gram of our test food, watermelon, and placed it in the mortar. We used our pestle to grind the melon then added five milliliters of distilled water to the mortar and continued grinding. Once the mixture was consistent, we added another twenty five milliliters of distilled water to the mortar and mixed it completely. We then used our transfer pipette with a clean tip to move fifty microliters of the newly formed mixture to a new test tube containing five hundred microliters of InstaGene Matrix and labeled it as our “Test”. We vortexed the tube to stir all of the contents.
Next, we placed our two tubes in a water bath that was set at ninety five degrees celsius for five minutes while we cleaned our lab area. After the five minutes finished, we placed our tubes in a centrifuge for five minutes at the highest speed. We left our tubes overnight in a refrigerator so that we could start where we left off in our next lab session.
In order to prepare for our PCR lab, we needed to create six different PCR tubes with the controls and our test food. We labeled six PCR tubes with our group number and assigned them each a number, one through six. Each of the odd numbered tubes received twenty microliters of a green plant master mix. The even numbered tubes got twenty microliters of a red GMO master mix. We put twenty microliters of the non-GMO control food in tubes one and two, and twenty microliters of a GMO positive control in test tubes five and six. We put twenty microliters of our test food into tubes three and four. We made sure to change out the tip on our pipette after every use to avoid any cross contamination between the DNA of our foods. Once all tubes were full, we put them into the thermal cycler.
We began day two of our lab by preparing a PCR gel. To create the gel, we put one and a half grams of agarose powder in a two hundred and fifty milliliter Erlenmeyer flask labeled with our group number. Next, we filled a fifty milliliter beaker with fifty milliliters of TAE buffer and our instructor added GelRed stain to the beaker. We added the buffer mixture to our flask and mixed to form a foggy liquid. We covered our flask with plastic wrap and put it in the microwave for thirty seconds. After thirty seconds, we used a heat pad to remove the flask from the microwave and gently swirl. We put it in for another thirty seconds and swirled it again, but it was not completely clear yet, so we had to put it in again for an extra fifteen seconds. After swirling the clear mixture again, we allowed it to cool for about five minutes, until it was cool enough to hold comfortably. We grabbed a box of pipette tubes to use in case we needed to pop any bubbles in the gel. Before pouring our gel, we had to set up the mold and place the comb in it to create the holes that we would later fill with our test liquids. We used the level to center our stand and had to place paper towels under the stand until it was perfectly level. My lab partner poured as much of the gel as he could into the mold and we set a timer for ten minutes so that the gel could cool completely.
Once the gel had cooled, we removed the comb and carefully took the tray out of the stand. Next, we put our tray in the DNA electrophoresis chamber with the sample holes facing the black side of the chamber. We filled the electrophoresis chamber with the TAE buffer until we reached the maximum fill line. We then put twenty microliters of the PCR products that we created in the gel wells very carefully. We set the power source at one hundred volts and began the PCR process. We set another timer for thirty minutes and waited for the DNA movement to finish.
After the thirty minutes ended, we brought the gel to our professor so that we could view our results through the lens of a machine and record our data. This completed our lab, so we then cleaned up our materials and gathered our findings.
Our results were invalid because only two of our lines that were supposed to be visible showed up properly. Even our control substances were not triggered as positive, so we can not determine through this experiment whether the watermelon that we tested is genetically modified. The image below shows our gel through the lens, so that all of the DNA lines are visible.
The top line is blank, as it should be because we left that well empty. The second line is our ruler and should have a mark at each measurement, but it does not. The next line down should have a mark at two hundred because it is GMO positive as well as the line after that, which should have a mark at five hundred. The fifth and sixth lines are our test food, which gave blurry results at the far right that are invalid. The last two wells contained a non-GMO control and did not show up at all on the gel.
We hypothesized that the watermelon must be genetically modified, but we did not have valid results to support our theory, therefore in order to prove our predictions to be true, we would need to do the experiment again. My group and I agree that the invalid results may be because watermelon is already a very hydrated food, and may have been too diluted to test. We may have made another mistake while pouring our gel, because some of it cooled while pouring which prevented us from using all of the gel. However, while the rest of our class was gathering their results, none of the test provided clear results. There must have been an error with one of our machines or our procedure that kept us from obtaining accurate findings to report.