We began our laboratory work on December 2, 2010 at 8:00 a.m. The head of the laboratory, Michael Gadermaier, gave a presentation on the basics of laboratory work. Our goal was to determine whether certain point mutations in the gene TAS2R38 correlate to the differences in our testers' taste perception of bitter substances. To do so, we examined the three single nucleotide polymorphisms (SNPs) on the DNA, i.e. the changes of single bases in the DNA. For each possible position of the mutation, we had to examine the presence of each mutation using the Real Time PCR Machine (qPCR), which amplified the individual DNA strands that we examined. The machine measured the brightness of each sample, which comes from the previously added probes. Probes are synthetically-created DNA fragments that are complementary to the DNA sequence that we studied. The probes for the wild type and mutants have different colors. We were able to determine which variant of the DNA that we investigated is present depending on which dye is released. The qPCR sends the measured data to a special computer software, where they are evaluated and can be plotted. For example, when a mutant probe is not bound, the software receives transmitted information from the qPCR that no luminiscence of the dye could be found. The software displays that no mutation was present in this sample. After all of the questions were answered, we went to the laboratory to put the practical information into practice.

In groups of three with three DNA samples each, we began to prepare the Eppendorf tubes, lovingly called Eppis. Each sample required three Eppis. Following our concept of anonymity, the samples were given a four-digit code, with which the Eppis were tagged as well. Thus, the results of the DNA analysis could be assigned later during the evaluation of the questionnaire.

In the first Eppi, we used 200 µl lysis buffer and a cotton bud. We cut off the unused plastic handle of the cotton bud with scissors that had been previously dipped in ethanol and sterilized over a Bunsen burner. The lysis buffer ruptures the cell membrane, which releases DNA, protein and the like from the oral mucosa cells. To aid this process, the Eppendorf tube was tightly sealed and placed in a heating block, where the solution was constantly shaken for thirty minutes at a temperature of 56°C. Then, we put the tube with the lid on the work surface, because the cotton ball had to be removed from the top. Next, we poked a needle that had been made sterile with a Bunsen burner, through the top of the Eppendorf tube. After each run, we had to sterilize the needle again in order to avoid contaminating the tubes with 'foreign' DNA. Then we put on the second, empty Eppendorf tube, which had been marked by the appropriate code. The two successive tubes were centrifuged for a few seconds in order to 'hurl' the solution into the empty Eppi. In this solution, the DNA, proteins and some other cell organelles were dissolved. To remove unwanted components from the solution, 100 µl of protein precipitation solution was added, which mainly destroys proteins that were still in the solution. After this sample had been chilled for five minutes, we put it into the centrifuge with the lid on the outside. Thus, the precipitated proteins were collected at the bottom of the tube. After the centrifugation, we saw a white pellet, which was the precipitated proteins as well as membranes and cell organelles. At this point, we were able to complete a targeted separation of the DNA by emptying the solution in the third, free Eppendorf tube while making sure that the pellet remained. After we finished this step, we slowly added 400 µl of iso-propanol. Then, each sample was tipped twenty times by hand. After precipitating for two minutes at room temperature in the laboratory, they were centrifuged for another five minutes. This guaranteed the precipitation of the DNA. The resulting supernatant was again removed and 500 µl of Ethanol was added with a pipette in order to wash the DNA. The samples were again centrifuged for five minutes. After this process, most of the pellets are no longer visible. We had to be cautious in this step to avoid shaking the samples. Even when the pellet is no longer visible to the naked eye, it still exists, and contains the necessary DNA. The supernatant was carefully removed; the remainder of the supernatant had to be gently with a pipette. It was imperative to ensure that the pellet remained in the Eppendorf tube. Through the centrifugal forces in the centrifuge, the DNA had accumulated in the pointed end of the Eppis, actually on the side of the cover. The pellet, therefore, was not directly on the top, but had been pushed to the edge, because the Eppi had been slanted in the centrifuge. Because the side with the cover was supposed to be shown on the outside, the DNA was transferred to this edge. We had to position the tip of the pipette on the exact opposite side and suck in with extreme caution, so that the pellet didn't get sucked out and emptied. To get to the really pure DNA, the Eppi was put again in the heating block for about 15 minutes at 56°C, in order to evaporate anything left over.

At the end, we added 20 µl of hydration solution. Thus, the DNA was in a solution, which did not contain anything that would distort the results. This solution is really a professional substitute for distilled water.

The samples were cooled, in case the qPCR was not immediately available. Then, the head of the laboratory, Michael, mixed our finished, isolated DNA with a master mix, consisting of probes, polymerase, nucleotides, primers and buffer. The mix was wrapped in a heat-sealed foil and put into the Real Time PCR. The foil prevents the samples from evaporating at high temperatures in the qPCR machine. After 50 cycles and about two hours, the Real Time PCR machine was finished and had transmitted its measurements to the program. The raw data was presented clearly to us and we were able to process the results.