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Week 4 Mid-term presentation week

             This week turned out to be a relative slow week, compared to my last few weeks at CMOP. Last week, Ben and I prepared Iwaco Harbor and main channel station # 7 prebloom/bloom/postbloom dinoflagellate 18S rDNA samples, and transformed the E. coli cells with these obtained dinoflagellate DNA. These E. coli cells were then plated and allowed to grow. Afterwards, Ben and I picked 10 colonies out of these plates and performed plasmid isolation on these picked colonies. The isolated plasmids from each sample were sent out to be sequenced, so we could use the DNA sequences of the dinoflagellate 18S rDNAs to further figure out the identity of these different dinoflagellate species present in the different times during M. Rubra bloom formation.

            We sent out samples to get sequenced, and Monday we got our “sequenced” results back. All 40 of the DNA sequenced results were absolutely terrible. The normal length of a dinoflagellate 18S rDNA sequence is around 1.5 kb. The “sequences” I got back, some were 170 bases and some were around 500 bases; the sequences the sequenced over a 1 kb were absolutely terrible and the DNA sequences were most likely wrong. I spent all day trying to analyze a sample that would give me some kind of results, but all of the sequences were too terrible that I wouldn’t be able to do anything to it, unless I send the DNA samples back to get “re-sequenced”. I was also not able to re-sequence my DNA samples again this week, because the sequencing specialist at the ONPRC was on vacation in China. L I got over this unfortunate event very fast, and started extracting more DNA from the water samples Ben and I collected last Friday 7/6/2012 throughout the CRE. These water samples, will be considered PREBLOOM water samples 7/6/2012, since they were collected and analyzed prior to the M. Rubra bloom. We went to 6 stations total on Friday (Iwaco Harbor, Baker bay, Young’s bay, Merts, Chinook and Hammond). We prepared the first step of extraction, which is cutting up the filter paper full of water extracts and incubating the filter papers in DNA extraction buffer. I was also able to finish my mid-term presentation slides that afternoon too (#productivity).

            Tuesday, DNA extraction experiments were performed on the 6 filter samples that were incubated in buffer on Monday. The amount of DNA obtained from the DNA extraction was not too bad, but not great either, indicating that there must be some level of impurity still present in the extracted DNA samples. I performed a teleaulex 18S rDNA PCR (a cryptophyte that M. Rubra specifically preys on). The teleaulex 18S PCR product was then run on a gel. Results were interesting, because Iwaco Harbor was the only place that didn’t seem to have any teleaulex present. The rest of the 5 stations did show to have teleaulex, while the Chinook station showed to have the most amount of teleaulex present. Now, time to raise questions. Even though Iwaco Harbor sample didn’t seem to show any teleaulex, does this mean that there really is no teleaulex cyptophyte present, or instead the Iwaco Harbor DNA sample was just not good? If Iwaco Harbor didn’t have any teleaulex, then does that mean the M. rubra bloom most likely won’t form there because the prey source isn’t available? However, the bloom starts every year in Baker bay and around Iwaco Harbor, does my result even make sense? Does this also mean Chinook will most likely have a chance of greater bloom formation?

            On Wednesday, Ben was extremely sick and was unable to come to work. I was asked by Peter to run a general 18S control PCR, to make sure that the extracted DNA samples from the 6 stations were actually good quality DNA and were able to produce good quality PCR products. I ran this PCR experiment again for the 6 samples, and the results were pleasing. All extracted DNA samples were PCR quality à good quality DNA. Surprisingly, Iwaco Harbor sample showed to have a lot of other cryptophytes, but not teleaulex. These results confirmed with the microscopic results that Ben found out previously. He found that Iwaco Harbor sample contained a lot of big cryptophytes, which were not teleaulex. I also looked at the Chinook and Iwaco Harbor lugols samples (from 7/6/2012) with Pete and Olivia. Since we are not “identifying mysterious species” expert, we couldn’t find any teleaulex in the Chinook sample even when it is supposed to contain the most amount of that species.

            On Thursday, I decided to perform a dinoflagellate 18S PCR on the 6 samples collected on 7/6/2012 (I did telezulex 18S PCR on Tuesday). By doing dinoflagellate 18S PCR on these 6 samples, I can identify which station has the most amount of dinoflagellate, and can selectively chose which station’s sample to sequence the dinoflagellate DNAs. After the PCR, I ran an electrophoresis gel for the 6 samples to see which has the most dinoflagellates (brightest DNA band in the gel). It seems like Young’s bay and Mert’s station have the most amount of dinoflagellate present. Ben and I decided to further analyze the Iwaco Harbor and Young’s bay sample. I did a gel extraction of the PCR DNA products, ligated the PCR products into a TOPO vector, transformed E. coli cells with these ligated vectors, and plated these transformed cells on agar plates to grow. All these methods are very common in sequencing DNA from artificial plasmids. I plated two different volumes of cells onto the plates to make sure even spreading of the cells (50 uL and 75 uL). Now, I can wait until Friday to check the growth of the cells.

            Friday is my day to present my mid-term presentation about my dinoflagellate project. It was very interesting hearing about all my fellow intern’s projects. I sort of get more of an understanding of what they do and what they are specialized in. CMOP does have a wide spectrum of researches, in all kinds of fields like biology, physical chemistry, chemistry, oceanography, bioinformatics etc. After my presentation, Ben gave me some constructive comments on my presentation. I’m used to giving very easy understanding presentation to a large group of people, and I definitely made my presentation very understandable. The thing is I wasn’t scientific enough, such as I would refer to things that have a scientific name, “cells” as “stuff”. I realized I was not very scientific in my presentation, so I know I definitely need to improve that for my final presentation. I’m very glad though, overall that most interns understood my project very well, and Ben said I was clear to the point. After the mid-term presentation, it is time to get data/results for my project and have a better idea of how to present my future results in the final presentation J