HoughtonlakeBoard.org

TIPS & EXPERT ADVICE ON ESSAYS, PAPERS & COLLEGE APPLICATIONS

Introduction Phenylthiocarbamide, or PTC, is a chemical with a very bitter taste. Arthur Fox, who synthesized the PTC, could not taste the chemical when some of the chemical’s dust escaped into the air. However, his lab mate complained of the bitter taste. With this observation, he concluded that the ability to taste the PTC is genetic and is variable among the human population. The TAS2R38 gene is responsible for our receptiveness to the PTC chemical.The goal of this experiment was to determine our genotype for the TAS2R38 gene. When conducting this experiment, a PTC test strip was tasted. Depending on the response to the bitter taste, which include a strong bitter taste, a mild bitter taste, or no taste of the PTC, phenotypes were determined. A strong bitter taste indicates the individual is homozygous dominant for the TAS2R38 gene. The inability to taste the PTC indicates a homozygous recessive genotype for the gene, and a mild bitter taste indicates a heterozygous genotype for the gene. Using cheek cells, a sample of DNA was taken from each person. The DNA was then separated  using polymerase chain reaction, or PCR. During the process, DNA is heated to split apart the two strands in a thermocycler.  An enzyme, Taq polymerase, then builds two new strands of DNA, using the two that were split apart as templates. As a result, there are now two DNA molecules, both with one original and new strand of DNA. This process is repeated by the fluctuation of the temperature in the thermocycler, allowing for the DNA to denature and synthesize (Ebmeier et al., 2006). Restriction enzymes were then used to cut the DNA into smaller pieces, and the samples were dyed and loaded into the gels for electrophoresis. Gel electrophoresis is used to separate the DNA using an electric charge. DNA is negatively charged, so applying a negative charge to the side of the DNA will cause the DNA to run towards the positively charged side of the gel. Smaller molecules of DNA will run faster through the gel than the larger DNA molecules. Therefore, the smaller DNA molecules will be closer to the positive electrode. The position of the DNA in the gel can be measured by the DNA ladder in the first lane. When reading the positions of the DNA molecules to determine whether the genotype is homozygous dominant or homozygous recessive, the ladder in the first row is used. A heterozygous genotype will show two separate marks in the gel. This is due to the two different alleles present in the genotype (Urry, 2014). Our study demonstrates the application of the Hardy Weinberg equilibrium. Hardy Weinberg has five conditions, including a large population size, no natural selection, no immigration or emigration, random mating, and no change in allele frequency due to mutations (Urry, 2014). All these requirements were met with the sampling of the students enrolled in Bio 124 this term. The sample population given is large enough, and meets all the requirements for the Hardy Weinberg equilibrium (Abecasis et al., 2005).The null hypothesis for this experiment is that the population will be in Hardy- Weinberg equilibrium. If the population size is in hardy- weinberg equilibrium, the null hypothesis will be accepted. However, the null hypothesis may be rejected as a result of the evolution of the human population. Methods: First, one must determine their predicted genotype based off their individual observations of how the PTC tasted. TT represents homozygous dominant, Tt is heterozygous, and tt is homozygous recessive. After each member labeled a 1.5ml Eppendorf tube with their name and group number and transfer 1000µl of the saline solution into their tube. They need to swab the inside of their cheeks and transfer the cheek cells from the swab into the Eppendorf tube. Then, each of the tubes must be placed into a balanced microcentrifuge and spun for 90 seconds at full speed. After that, label another 1.5ml tube with the same things from earlier and set it aside. Then, empty the tubes in the microcentrifuge of its liquid without disturbing the pellet that should’ve formed at the bottom. Once the micropipette has been set to 30 µL, resuspension of the cell can take place and the transfer of the 30µL of cells to the empty 1.5mL tube can begin. To resuspend the beads, invert the Chelex solution and transfer 100µL of the solution into the microcentrifuge tube. Remember to sterilize the needle with an alcohol pad and create a hole at the top of the tube before placing it in a dry bath for 10 minutes at 99°C. Once it has boiled, shake it for 5 seconds and place it into a balanced microcentrifuge for 90 seconds. Then, label a new tube and insert 30µL of the clear liquid only and store it in ice. Next, label the side of a PCR tube with the number assigned and without extracting any beads insert 22.5 µL of PTC primer into the tube and leave it to dissolve. Once the solution is a pinkish purple, add 2.5µL of cheek cells into the tube containing the PTC primer. Store it in ice and bring it to the TA to be placed in a thermal cycler. After the programmed 30 cycles, the TA will store it at -20°C until the next lab. Next, to make the 2% Agarose Gel, pour the pre-weighed 1.2g of agarose powder into a 250mL flask. In a graduated cylinder add about 60mL of TAE buffer and transfer half of the buffer into the flask with agarose powder. The mixture must be swirled for about 20 seconds before the rest of the buffer can be added into the flask. Which will be swirled a couple more times before placing into the microwave for 45 seconds. The swirling and microwaving process will be repeated once more to completely dissolve the powder. Once dissolved, allow it to cool on the the bench before adding 6µL GelRed stain. Then, place the gel rig to the side and wait for the TA to check the setup of the gel and comb. Once your TA gives the signal, pour the mixture onto the gel and allow it to solidify for about 30-40 minutes. After about 30-40 minutes, lay a piece of saran wrap on the table and place the gel onto the wrap only if the TA instructs you to do so. From there proceed to wrap the gel in the saran wrap, which will then, be wrapped in tin foil and taped with your section and group number. First, determine the number assigned to each member from the previous lab and label two 1.5mL tubes with one marked with “U” and the other with “D” and the assigned number. In each set of “U” and “D” tube make a side with one’s initials. From there transfer 10µL of each individual PCR product to the “U” tube and place it into the ice. Then, add the remainder of the PCR solution into the “D” tube and the TA will add the 1 µL of  HaeIII to the solution. Next, mix the “D” tube in a microcentrifuge by pulsing it and then, placing it in a dry bath at 37°C for 20 minutes before putting it in ice. Once each member has finished practicing loading a gel, unwrap the gel and place it in the gel rig. When the TA states the rig is oriented correctly; begin to fill the chambers with 1X TAE electrophoresis buffer. Next, transfer 20 µL of pBR322 size marker into lane #2 and have each member insert 10 µL of the solution in the “U” tube and about 15 µL of the solution in the “D” tube into the wells after lane #2. Then, in the tube labeled “100 bp Ladder” containing 10µL of 100 bp ladder and 4µL of loading dye transfer it into lane #11. The TA will set the power to 150V for about 45 minutes

Post Author: admin

x

Hi!
I'm Irvin!

Would you like to get a custom essay? How about receiving a customized one?

Check it out