Spring 2003

Introduction

This lab will use samples of microbial mat that were collected in Yellowstone National Park by Dr. Boomer. The template genomic DNA will be extracted/isolated from the Yellowstone samples. Then a specific gene will be amplified from the genomic DNA by using PCR. After PCR has been performed the goal is to have amplified a target 16S rRNA gene sequence. This sequence can be used as a fingerprint that enables comparison and identification of our sample bacteria population.

	Genomic Methods and Results
Methods: Sodium acetate (3M) and 95% ethanol were added to the samples. The samples were then put into the freezer (shown to right) for 20 minutes at -65 degrees Celsius. This step was to allow the DNA to precipitate out of solution so that it could be pelleted through centrifugation, and then resuspended back into solution, incubated, and amplified through PCR.
Results: The picture to the right shows the phenol bubble that was present in the sample after extraction. Due to the phenol still remaining in the sample they were unable to be used for PCR amplification. Dr. Boomer and Danny gave us new DNA samples to be used for PCR. If this part of the lab had worked no phenol bubble would be visible and a DNA precipitate would be visible.

PCR Methods and Results

Methods: This is a picture of the PCR apparatus. It performs all of the cooling and heating cycles necessary to duplicate the DNA

Results:

Primer = forward and reverse for the type indicated (Red or 16S).

Lane 1: buffer C + DNA + Red Primer

Lane 2: buffer G + DNA + Red Primer

Lane 3: buffer E + DNA + Red Primer

Lane 4: buffer I + DNA + Red primer

Lane 5: buffer K + DNA + Red primer.

Lane 6: buffer C + DNA + 16S primer

Lane 7: buffer G + DNA + 16S primer

Lane 8: buffer E + DNA + 16S primer

Lane 9: buffer I + DNA + 16S primer

Lane 10: buffer K + DNA + 16S primer

Lane 11: Molecular weight standard

Lane 12: Empty

Discussion

The picture of the gel shows the primer band in each Lane #1 through #10 as the top band. The molecular weight standard is used to determine the approximate weight of the genomic DNA that was targeted for amplification through the use of specific primers. For this lab the most significant bands on the molecular weight standard lane are the two bottom bands that are of the 500 and 2000 molecular weight range. Lane #1 through #5 show only primer and no red 16S rRNA being amplified. Lane #6 shows primer and amplified rRNA, but the amplification was not tremendously successful. Lane #7 through # 10 show primer and amplified 16S rRNA present. These banding patterns are in the 1500 range. This range is where this type of organism’s gene product should show up.

The methods for extraction of the DNA of the Red and 16S bacteria were flawed in some way. However, the reason that phenol remained in the samples after the DNA had been extracted is unknown. Phenol/Chloroform is an organic solvent that forms a distinct lower layer when mixed with aqueous liquids which allows for the extraction of most cell parts (including most proteins, membranes and pigments), leaving primarily DNA in the upper layer. Since there was phenol still in the sample, this implied that other cell parts besides DNA were also present in the sample (or other metals, acids, and bases possibly from the organisms environment), making then unusable for PCR. It could have been due to the phenol/chloroform not sitting long enough before they were added to the samples causing the phenol and chloroform not to be fully separated.

PCR was used to amplify the DNA of the organism and then it was ran on a gel (by Dr. Boomer and Danny), soaked in ethidium bromide, and then exposed to UV light while photographed. This resulted in the photograph shown in the results section of PCR methods and results. The PCR for 16S was successful, but the Red was not. There is no way to pin point the exact reason why one sample prepared in the same way would work and the other would not. PCR is notorious for working only some of the time. In the grand scheme of things this lab amplified DNA that had been extracted/isolated from the microbial mat sample through the use of PCR. This DNA genome can then be used as a template for amplification of a particular rRNA sequence that can be used in later labs as a fingerprint sequence allowing the comparison of our bacteria sequence to other bacteria sequences and eventually identification of our bacteria samples.