Delonna White delonna@vims.edu (804) 684-7458, 7337 Chesapeake Bay Hall,  N114

Hello everyone, my name is Delonna White and I am a first year grad student at the Virginia Institute of Marine Science. Over the summer, I had the opportunity to work under the guidance of Nancy Stokes, who is the technician for the Oyster Disease Lab, who helped me start my preliminary research on a water fungus known as Sirolpidium zoophthorum. With the help of my advisor, Dr. Eugene Burreson, I was able to work on my project using a grant from the National Oceanic and Atmospheric Administration (NOAA).

Several strains of S. zoophthorum were shipped to me on the 11th of June of 2004 from the Milford Laboratory, and DNA extraction, quantification, and amplification through PCR were performed over the span of three weeks to determine if the extracted fungal DNA would amplify using the chosen primer sets: COII-R, COII-F, LSU-0025F, LSU-1170R, LR6-O. From this first experiment, I concluded that all three types of primer sets were efficient at amplifying the genetic material of the fungus. Therefore, I was able to conclude that the sequence of these primers was present in the fungal DNA.

Gel picture of the extracted DNA shows that 1µL of DNA is just as efficient at amplifying the genetic material as 4 µL, and that both extraction kits work well on this particular DNA.

Gel picture of the first PCR product using the primers COII-R, COII-F, LSU-0025F, and LR6-O on the DNA extracted from the DNeasy Plant kit and the QIA amp DNeasy kit shows the LSU/LR6-O primers produce stronger Bands than the COII primers. Amplified DNA was not collected for the LR6-O primers using sample A from the QIA amp kit, and is therefore not shown on the gel.

Based on the results from the first PCR products, I predicted that the LSU-0025F, LSU-1170R, and the LR6-O primers would be the best candidates for developing the DNA probes for the Sirolpidium fungus because their amplification products show up as stronger bands on the gel, which means there is more product to work with.

The next phase of my project was to obtain cultured samples of Sirolpidium zoophthorum to ensure only one fungal species' DNA was being tested. If cultured samples were not obtained, inconsistencies in the data might develop in the future.

The cultured strains of S. zoophthorum arrived on August 4th of 2004. Extraction was performed this time using just the QIAamp kit, and PCR was later performed on the sample using the following primer sets: COII-R/ COII-F and LSU-0025F/ LSU-1170R.

For this PCR reaction, 3 reactions were made for each primer set, which allowed me to get a triplicate amplification. This was necessary so that I could lower the chance of getting polymorphisms when I am ready to ligate the PCR product into the plasmid DNA.

Gel picture showing the triplicate amplification products of the two primer sets.

Once the PCR products were retrieved, the next step of the process was to purify the PCR amplified DNA, which was done using the QIA quick PCR purification kit protocol. Once this was completed, the TOPO cloning reaction was used to insert the PCR products into the plasmid vector. After the TOPO cloning reaction was made, competent cells, or bacterial cells that have spaces in their walls for DNA to enter, were gently mixed with DNA from each primer reaction. After the completion of the reaction, plates were spread using 25 µL and 50µL of each primer set and allowed to incubate overnight, and then placed in the refrigerator the following morning.

Colonies were observed for the successful transmission of the fungal DNA into the bacterial host DNA. Successful colonies were picked to be re-streaked to verify that they did in fact contain the fungal DNA. Six successful re-streaked colonies were chosen from each primer set (COII primers and the LSU primers), and a Phenol Chlorophorum extraction was set up to lyse the plasmid DNA from the E. coli bacterium using the protocol for Quickscreen DNA preps. The lysed plasmid DNA was then run on a 1.5% agarose gel.

This gel picture shows the lysed plasmid DNA from the 6 re-streaks. The Negative controls were run on each side of the plasmid DNA and represent unsuccessful fungal DNA inserts. The first band on the gel represents the bacterial DNA, or genomic DNA. The second band represents plasmid DNA.

The gel confirms that each of the 12 bacterial clones contained the correct fungal inserts.

The next process of the experiment consisted of preserving the DNA so that it could be used in the future to decipher the sequence of the fungal strain in question.

Major advisor: Dr. Eugene Burreson