Who Is Diseased? No one... That we can tell

Our lab took place over three days. The first day consisted of us making our samples with our own cheek cells, after collecting them in flip-top tubes, we then put them into water baths at 56 degrees Celsius for five minutes, vortexed them, then placed them back in the water bath for a remaining five minutes. After finishing the designated ten minutes, we transported the samples to a 100 degree bath. After five minutes we centrifuged and then put them in the refrigerator until the next day.

The second day of the lab we performed PCR amplification. We retrieved our samples from the refrigerator and then we transferred 20 micro liters of the DNA template from the screw cap tube into the bottom of the PCR tube. We were very careful in not transferring any of the matrix beads because they would have killed the DNA polymerase. We added the yellow master mix to each of our samples and mixed well. Then we added our PCR tubes into the thermal cycler, where it underwent 40 cycles of PCR amplification. 


On the third day, we retrieved our tubes and attempted at performing gel electrophoresis. We filled ten lanes of the gel, the first four with "controls" used to compare our results with. The second four lanes had our samples in them. But, unfortunately when we were adding our results one of the samples was being added when the bottom of the gel was punctured and the sample was flooded underneath the gel. Unfortunately, when we got our results back none of our samples showed up except the controls and my own sample (which was the first loaded). So we could not compare our results to our controls to find out who had this diseased gene. However our one sample that did show up, my own, was heterozygous. The way we know this was because after the gel electrophoresis was performed my sample and our heterozygous (+/-) control lined up with my sample. 


Although our results did not all show up, we did learn how to test for certain genes and I was able to see how this process could be important to scientists and other people trying to compare information in different genes. 

Who Is Diseased?

In this lab, we will be using PCR to examine our own DNA sequence in a test tube. We will be looking for a particular piece of DNA that is present in most people, but not all people. We will be determining this through PCR, which has begun transforming molecular biology into a multidisciplinary research field. On the first day we will be extracting DNA from our cheek cells. We will break open the cell membranes by putting the samples into a water bath, then we will use instagene matrix beads to kill DNase. On day two we will perform PCR in three steps, as shown below....

In this lab PCR has three main steps. The first is denaturation, the second is an annealing step, and the third, an extension step. In the first step, the DNA seperates into two single stranded molecules. The templates must be sperated before the polymerase can generate a copy.
During the annealing step the oligonucleotide primers find their complementary sequenceds on two single stranded template strands of DNA. After they are annealed they can be used as primers for the Taq DNA polymerase. In extension Taq DNA polymerase's job is too add nucleotides (A,T, G and C) to the primer to create a complementary copy of the DNA template.

After running through this, on day three we will use gel eletrophoresis to diagram our results.