Copyright 2003

Introduction

We used this lab to isolate genomic DNA from unstudied samples of hot-spring bacterial mats. Polymerase Chain Reaction (PCR) techniques were used to clone and amplify the gene that codes for the 16S ribosomal subunit. This particular gene is often useful for determining relatedness between different species of bacteria because of its conserved nature.

	Genomic Methods and Results
Methods: We centrifuged the samples after adding the phenol-chloroform and vortexing. This separation process was used to separate the cell debris from the genomic DNA
Results of sample 99: During the separation procedure, some of the phenol-chloroform was taken up during the removal of the aqueous layer. This left a “bubble” of phenol at the bottom of the microfuge tube. Since this bubble was where the genomic DNA sample was supposed to be, it would have been too difficult to separate the two in the time we had for lab. Therefore, in lieu of the genomic samples that were to be obtained during this procedure, we were given DNA samples that had already been purified.

PCR Methods and Results

Methods: Ten strip tubes were set-up as two different sets of salt-buffer gradients. One set had RED forward and reverse primers added to them (shown by the 5 leftmost tubes opposite). The other set had 16S forward and reverse primers added to them (shown by the 5 rightmost tubes opposite). Water and Taq polymerase were also added to all tubes as well as our sample DNA. The tubes were then put into the PCR machine.

Results of the Imperial ’00 sample:

Lane 1 (left side of picture opposite) was molecular weight standards. Lanes 2 through 6 represent the samples from the tubes that had the RED primers added to them. Lane 3 looks like it may have been positive for red sequences. Lane 5 is positive for PCR clones. Lanes 7 through 11 represent the samples from the tubes that had the 16S primers added to them. Lane 8 was the lane that is positive for PCR clones.

Discussion

Since there were problems avoiding the phenol-chloroform layer during the extraction process we ruined the mat samples that we were given to perform genome isolation on. Due to this, we were given a different sample that already had the genomic DNA isolated. PCR was performed on this new sample. The results of the PCR were positive for clones made with both the Red and 16S primers. It was interesting though that Red clones were produced in a different buffer concentration than 16S clones. The results of the banding patterns were consistent with what we would expect to see. The band representing the Red PCR products progressed further than the band representing the 16S products because the cloned Red sequences were shorter that the cloned 16S sequences.