John Koberstein's blog

I began the week with optimism that my Cerc5 primers would be usable for qPCR and that I could get some numerical results confirming it is located in just the Columbia River Estuary. This plan did not work out as my first qPCR run featured significant amounts of contamination in the no template control. This confirmed once and for all that my primers must be contaminated and that I needed to order a new batch. Until the new primers arrive I have begun working on quantifying the distribution of a new protist organism- euduboscquella.

By chance I discovered one of the primer sets, Cerc5, I had initially discounted actually works to selectively amplify the 28S USE. Even better this primer set seems to only amplify sequences found in the Columbia River Estuary. It so far has not been found in the Chesapeake, Amazon, Beaufort and Susquehanna samples I have tested. This is exactly what I have been trying to find- a CRE specific species of Cercozoa.

This week has been less succesfull than I had hoped. Every PCR reaction I run has amplified DNA in the control. This makes it impossible to get meaningful data. Something is contaminated with DNA but it has not been clear what reagent it is exactly. Until I find the source of the contamination I cannot proceed with project. Other than those troubles, I have been working on getting extended USE sequences for other Cercozoan species so that I may design more affective primers.

This week with Tuesday being a day off from work to tour the Bonneville Dam and Friday being the 4th of July only consisted of 3 days in the lab. I recieved a new batch of primers on Monday that were designed to amplify USE from Cercozoa isolated in Vancouver. So Wednesday and Thursday I ran a few PCR reactions on samples from the CRE as well as La Push as a potential positive control. I was hoping La Push, the closest total extract I have to Vancouver Island, would contain some of the same Cercozoa but unfortunately the results of PCR indicate that it does not.

After recieving the sequence information of my two plasmids, I found both to match the database sequence I was attempting to amplify. Knowing the number of basepairs in the sequence then allows for the calculation of the number of copies of the sequence in a known volume of solution. This is necessary information to run qPCR on a sequence as standards are required to create a concentration curve for which unknown samples can be compared to in order to quantify the presence of a gene.

After recieving an order of primers I am now able to work on amplifying the USE of some of the Cercozoa organisms I am studying. The initial PCR experiments I ran were attempts to amplify DNA from three 28S unique sequence elements that closely matched species in the Bodomorpha and Gyromitus genera as well as an unclassified Cercozoa species. The Bodomorpha primer appeared to amplify LSU sequences with little specificity as evidenced by a smear of DNA visible after running a gel of products.

I am John Koberstein, an undergraduate intern with CMOP, and will be studying the distribution and abundance of Cercozoan species in the Columbia River Estuary. This being the first week of the internship I have had to do a bit of learning. I am working under Dr. Peter Zuber and a research technician Ian Vorhees. To get acquainted with theories regarding microbial biogeography and dispersal Dr. Zuber has given me some reading to do from the current scientific literature.