Tas2r38 Locus Lab Summary

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

In order to determine my genotype, DNA was extracted from the cheek (buccal) cells. After placing the extracted DNA into the Nano drop, the concentration of the sample was observed in order to determine if it would be successful in the polymerase chain reaction. The sample was then placed into a spectrometer to quantify the amount and purity.

Genomic DNA is heterogenrous because it shows 2 fragments on 2% agarose gel which come from parents, mom and dad. Moreover, the tandem repeats(n) is within the standard limit (14-41) of heterogenic DNA. So, the sample is heteregenerous.

For this experiment, we used two restriction enzymes called Ava II and Pvu II. Four tubes were needed for the experiment as well. Each microtubule was labeled accordingly, A, B, and C. Using Micropipettes, we added 2 microliters of pUC19 DNA into each tube. To make sure the DNA was on the bottom of the tube, we tapped each tube on the lab bench. Each tube had its own specific amount of different solutions added on, however the final volume for each tube was 30 microliters. Tube A had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Ava II enzyme. Tube B had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Pvu II enzyme. Tube C had, 23 microliters of DI Water, 3 microliters of the buffer, 1 microliter of Ava II, and 1 microliter of Pvu II. The last tube C had, 25 microliters of DI water, 3 microliter of the buffer, and no enzymes. Each solution was added according to how it is written here. After we finished adding the solutions to each tube, we tapped the tubes on the lab bench to make sure the

However, DNA fragments of 3 lab subjects didn’t show up on the gel. The allele

To determine the genotype of the members in my group and the class with regards to the TAS2R38 and LCT genes, first, DNA was extracted from epithelial cells to be used for PCR to amplify the DNA of the targeted loci. Then, restriction enzyme digestion was performed to cut the DNA into fragments in order to perform Restriction Length Polymorphism Analysis to decipher the different lengths. Differing lengths of the DNA accounts for different alleles on that specific gene, therefore determining heterozygosity and homozygosity of the genotype.

I hypothesize that there will be a strong deviation between the genotype frequencies of the two lab sections combined and that of the US. This experiment consisted of 26 individuals, of varying ethnical backgrounds, ages, and genders, who amplified their own DNA through the use of PCR. Their DNA was examined for the presence [(+/+) or (+/-)] or absence [(-/-)] of the Alu insertion through the use of gel electrophoresis.

If there is smeared DNA bands then the DNA was degraded and the student would want to avoid the nuclease contamination. Lastly, if the student sees anomalies DNA band migration then the improper electrophoresis conditions were used. When conducting this experiment be very careful as to how much DNA or electrophoresis gel and as well as voltage

The columns were centrifuged at 15,000 x g for 1 minutes to remove the supernatant. 0.5 mL of Buffer QG was added to each column to remove trace amount of agarose, and 0.75 mL of Buffer PE was added to wash the columns for 5 minutes. Finally, the DNA was eluted with 35 µL of Buffer EB into a clean microcentrifuge tube. The elution buffer was allowed to soak in the membrane for 3 minutes before being centrifuged. Eluted DNA samples were stored at -20

The gel was covered with a buffer and then six samples labeled A-F were deposited into the wells using a micropipette. Three of these samples (A, B, C) were control samples to compare to D, E, and F (the mother, child, and father). The safety cover was placed on the unit and then brought to a power source. The leads were connected the chamber and left for approximately 20 minutes. The agarose gel was removed from the tray and placed onto a sheet of plastic wrap. An Ethidium Bromide card was placed face down onto the gel to stain for approximately 5 minutes. Finally, the card was removed and the gel was placed on top of a UV light. The samples were pushed towards the center due to opposite electric charges. Agarose gel separates the DNA samples by the way they were cut. The restriction enzyme MST II cuts the DNA strand at CC/TNAGG where N is any nucleotide base. If the enzyme recognizes this, it is cut. If it does not recognize it then the strand is left whole. We were able to observe the DNA strands due to them being dyed and placed over a UV light. The control samples were utilized so that the other samples could be compared to test for their genotype. The data was analyzed in this way to differentiate between the different genotypes and the number of bars they

Dr. Park had created the gel for the class. I added 10 μl of the 5x PV92 XC loading dye into the PCR sample. I added 10 μl of the MMR-DNA marker into lane one, 10 μl of the homozygous (+/+) into lane 2, 10 UL of the homozygous (-/-) into lane 3, 10 μl of heterozygous (+/-) into lane 4, and 20 μl of my sample into lane 5. After the other students loaded their sample into the gel, I hooked up the electrodes and turned on the electrophoresis apparatus, which was at 100 volts. The electrophoresis apparatus ran for 40 minutes, which was until the marker dye had traveled half of the distance to the end of the gel. I then removed the gel and submerged it in the Fast Blast DNA stain (100x) using the staining tray. To create the staining gel, we used the formula M_1 V_1= M_2 V_2. The initial concentration of the gel was 500x, but we needed 100 ml of a concentration of 100x. I calculated that we needed 80ml of H_2 O and 20 ml of the dye. I stained the gel for 2 minutes. I rinsed the gel in warm water for 10 minutes. I compared my sample lane with the control lane and recorded my

Next, an ice bath was made by filling up a beaker with ice, them filled it with about two-thirds of tap water. Then, ethanol and the DNA extraction was poured into separate medium-sized test tube and set in the ice bath. Then, six layers of cheesecloth was gathered and cut into square and was fitted into a large funnel. A large test tube was attached to a ring stand and set the funnel and cheesecloth into the top of the large test tube. Then, the strawberries were placed in a clean ziplock bag.

The recommendations before starting the lab are to conduct a practice experiment with inserting the DNA with the micropipette. This decreases the chance of rupturing within the gel, and if this is to happen inaccurate results will be displayed. To begin the experiment, insert the gel into the electric chamber filled with THE solution so that it covers the agarose gel. Once the agarose gel is in place, take the Missouri DNA and extract from the sample tube with the micropipette and insert it into one of the wells. Repeat this process with each tube, but after inserting a DNA replace the disposable plastic tube with a new tube, so no inaccurate results will occur. After all six wells are filled, enclose the chamber and initiate the chamber with

Then 5µl of DNA samples were transferred into an individual Eppendorf tube containing the master mix solution. Once completed all 15 tubes were immediately transferred to a thermal cycler.

Each STR is polymorphic, however, the number of alleles is small. Typically each STR allele will be shared by around 5 - 20% of individuals. The power of STR analysis comes from looking at multiple STR loci simultaneously. The pattern of alleles can identify an individual quite accurately. Thus STR analysis provides an excellent identification tool. The more STR regions that are tested in an individual the more discriminating the test becomes.