Biology 475 Molecular Biology Lab One - Genomic Isolation and PCR Shanna Briggs Copyright 2003

Introduction The goal in this exercise was to isolate, and amplify genomic DNA from unstudied bacterial mat communities collected from Yellowstone National Park.

	Genomic Methods and Results
Methods: Following the first extraction with phenol/chloroform, the tube contents were centrifuged at full speed for 5 minutes. The DNA remained in the top layer.
Results: Despite our best efforts, the entire class had a phenol bubble mixed with our DNA. As a result, we were given new DNA samples to use for the PCR analysis. My new sample is labeled IMP- 2000.

	PCR Methods and Results
Methods: The cocktail we made for the PCR contained 66 ul water, 3 ul forward primer, 3 ul reverse primer, .5 ul Taq Pol and 3 ul DNA.
Results: Lane 1 is the standard markers. Each sample was amplified using 16S specific and Red specific primers in the buffers C, E, J, I and K. I loaded red first, with the buffers in order (lanes 2-5), skipped lanes 7and 8, and then loaded the 16S with the buffers in the same order (lanes 9-12).	1 2 3 4 5 6 7 8 9 10 11 12 13

Discussion

The technical aspects of this lab were the techniques used for isolation and the PCR. We were successful in releasing the bacterial contents, but ran into difficulty when trying to isolate the DNA because of the phenol used. Had we not had bubbles of phenol in each of the tubes, we would have dried the precipitated DNA product. This product would have been used for PCR amplification. As it was, we used new DNA samples for the PCR instead.

PCR is a sensitive procedure for amplifying small samples of DNA to a level where they can be useful for subsequent analysis. It mimics the process of in vivo replication. Taq Pol, a thermal stable polymerase, fulfills the role of the DNA Pol and the addition of manmade primers replaces the primers laid in vivo by RNA Pol. To unwind the helix PCR relies on heat instead of helicase and single stranded binding proteins. I was not successful in amplifying these samples, with the exception of Lane 12, so the results contribute very little to the overall project. PCR is an especially sensitive procedure so there are many reasons for these results. Most likely the error was my own, especially because I lacked experience with the tools used.

The overall significance to the project, had the amplification been successful, is that cloning and sequencing the 16S ribosomal DNA depends completely upon PCR technology. In order to assay the diversity of the red bacterial populations, there must be a method to produce a large enough quantity of DNA to be used.