Biology 475 Molecular Biology Lab One - Genomic Isolation and PCR Andy Mikles

Introduction As the title might allude to, the goal of this lab was to isolate DNA from bacterial mat communities collected from Yellowstone. Using several chemical and biological agents, a fairly clean sample of DNA from the bacteria can be isolated. The point of this isolation is to allow for PCR. PCR will amplify the genome of the bacteria culture we started with. Specifically it will copy the 16s gene of the bacteria since we used a primer specific to the sequence of that gene. With billions of identical copies of the 16s gene from a single bacteria, we can then study that bacteria’s individual genomic makeup.

	Genomic Methods and Results
Methods: turning the tubes upside-down in order to dry the pellet. This allows us to resuspend the pellet in water.
Results: It ended up that Phenol was still present in our samples. This is a recoverable mistake, but it was deemed too time consuming for lab purposes, so the decision was made to do different DNA samples for PCR.

	PCR Methods and Results
Methods: After adding DNA, buffers, Primers, and Polymerase into 10 different tubes, we put them on ice until they went into the PCR machine.
Results: my results from PCR show 3 samples that have distinct bands, and the one are the far right which is the weight standard. The two on the left are short lengths of amplified product, which are too short to be a good insert. The one on the right next to the M.W. standard is a pretty long sequence, just about right to make an insert out of.

Discussion

We studied sample number 99 from hillside # 2. we first added lysozyme to digest the cell wall, the we added EDTA, which inhibits the release of nucleases, which would digest the DNA if it were allowed to be released. We then added SDS in order to disrupt the cell membrane, letting EDTA in and lysing the cell membrane. We then performed extractions with phenol chloroform which precipitated the cell parts leaving the DNA in the supernatant. We remove the DNA from there, and precipitate it in order to get a clean sample of it. There is not much to see during these steps, you just have to add the chemicals and know that it is working. Unfortunately, ours did not. We had phenol left over in the tubes after extraction, and that means that there are cell parts are still in there, and the DNA, which is supposed to be at the bottom, is not. So we had to abort this part of the lab.

The second part of our lab was performing PCR. We added a combination of 5 different primers to 2 different buffers in order to get the right environment for our DNA to get amplified in. we then added our polymerase and DNA and cycled it in a PCR machine. In order to figure out which samples worked the best, they were ran in gels and photographed with UV light. The results are as stated above, which it seems that only one sample out of ten got a good result.