Blog #5: DNA Extraction and Amplification

Hello!

The last few weeks of my summer research have been dedicated to the third component of my project, analysis of bacterial community composition. This is done through T-RFLP, or terminal restriction fragment length polymorphism. In order to do this assay, I first had to extract DNA, and I am still in the process of making sure I can get good amplification so that I know I have enough DNA with which to work. I am leaving campus tomorrow to prepare for a family trip, so I probably won’t be able to do T-RFLP until I resume research in the fall. However, I have still gotten some exciting results despite some setbacks in this part of my project.

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Blog #4: Bacterial Production Assay and Epifluorescence Microscopy

Hi guys!

Here’s some additional information on how my project has been going. First, though, I would like to do a recap of what I am working on. There are three major components involved in acquiring data for my project: I used a tritiated leucine assay to determine bacterial production; I used epifluorescence microscopy to enumerate bacterial and viral abundance; and I am currently doing DNA extractions to prepare for T-RFLP assays, which I will use to determine bacterial community composition (this shows how diverse the bacteria in my microcosms from Lake Matoaka are after being exposed to different treatments). I will talk more about T-RFLP later, but here is a description of epifluorescence microscopy and the results from the tritiated leucine assay.

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Blog #3: Mid-summer Updates and Changes in My Project

Hi everyone! Hope you had an enjoyable 4th of July.

The last couple weeks have been especially busy. During week #4 of my research, I created treatments and incubated them in microcosms (2-L carboys) at room temperature. As I collected all the necessary subsamples, I began making slides from the frozen cryo vials. I also pooled the triplicates, or three replicates, of my treatments onto 0.22 micron nitrocellulose filters to capture bacteria, after which I stored the filters in the -80 ºC freezer (which is extremely cold!).  I will use these filters later when I conduct t-RFLP (terminal restriction fragment length polymorphism) to assess changes in bacterial community composition. Lastly, I helped Professor Williamson prepare vials with sub-samples from each of my treatments so they could be analyzed for bacterial production via tritiated leucine assays. Since tritiated leucine is radioactive and I am not certified to handle such substances, Professor Williamson was primarily responsible for this part of the project.

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Blog #2: Preparations – Timeline, Equipment, and Materials

This is a blog I had been writing last week, but I got so caught up in my trial run and project preparations that I never got around to posting it. Last week during the KCN trial run and yesterday during the set-up of my microcosms, I ran into some obstacles that required some modifications of my project, which I will explain later. Overall, though, filtration and set-up ran very efficiently, and my project is now underway!

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Blog #1 Literature Review and Protocol Preparation

Hello!

It has now been two weeks since I began my summer research in the Bacteriophage Ecology Lab at William & Mary. I have spent this time reviewing articles and books for ideas as I’ve prepared the details of my experimental protocol. I have also shadowed some lab techniques that I will be using for my experiment once I have collected samples and set up microcosms, which will be incubated in the lab. On Monday, June 6, I learned to use tangential flow filtration to obtain filtrates and concentrates with which to create treatments. Other lab members have shown me how to prepare slides for enumeration via epifluorescence microscopy. I will later explain these lab techniques in further detail.

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