Second Post!

Hello Friends!
I hope everyone’s summer has been going well. Here in the ISC I have been productive in synthesizing my new unnatural amino acid protyro. This UAA has the ability to “click” onto other molecules, meaning it has an alkyne group that is able to link to other alkynes, azides, etc. Creating protyro has been an adventure by itself. The actual synthesis is six steps, that involve a variety of organic reactions such as brominations, reductions, nitrations, and deprotections. I have attempted the synthesis twice now. The first time the reactions were going well until the second to last reaction, which was a malonic ester synthesis done via a microwave. When doing a microwave reaction, one needs to add a stir bar to allow a thorough microwaving, but I had forgotten about this fact. After finishing the microwaving, my professor informed me that I had forgot the stir bar, so I added the stir bar and re-microwaved. It is tough to know whether this is what caused the reaction to fail, but after I ran a column, I generated very little product. I decided to start over with precursor that I saved. This time I successfully completed (or thought I had successfully completed) all six steps. I tried to take a H NMR of the final product, and even though they say twelfth time is the charm, all twelve of my attempts were to no avail. This may be due to the fact that I had created very little product, or that the UAA was just difficult in general to read. I tried to NMR in various solvents: CDCL3, Methanol-D4, and even D2O. I decided to go ahead and insert protyro into GFP Waldo, a dimer GFP hybrid, where the UAA was inserted into the 151st residue. Despite having an “iffy” product, my confidence in this insertion lowered once our lab realized our comp cells were tainted. Comp cells are cells whose plasmids are easily edited, and this insertion and selection is done via inserting  antibiotic resistance, namely Ampicillin and Chloramphenicol. Without going into too much detail, we found antibiotics inherent in the bacteria, which means many of our bacteria colonies could be contaminated. Over the course of the next few days, my goal is to successfully synthesize protyro, which means having high yields for each of the synthesis steps.
My time with Florence the fluorimeter has been intriguing, yet tiresome. I have done over a hundred scans of different proteins with my AzoBenzene compound, many of these with varying solutions such as PBS buffer and 1X tris buffer. I have seen many interesting traits from the fluorimeter, but handling the data can be overwhelming. Each scan produces several hundred data points, all of which have to be transferred onto a separate excel sheet and compiled. While this is a very necessary part of my research, I have become weary of the constant scans, and I’m worried I have become inundated with graphs on lines.
I have much more information and results to come in the next few days so stay posted, and thanks for reading!

Zebra Finch Axons: Calcium Imaging and More!

Hello!

This is sadly my first blog post regarding actual lab work and data thus far. The delay has been caused by orienting myself with several new techniques, experiments, and culturing protocols that have been optimized this summer. Now that I am finally in the groove of things, I feel it necessary to share my experience!

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La Sauceda: A Case Study in Projects of Historical Memory in Cádiz

Since I left for Cádiz, the focus of my research project changed  from evaluating the efficacy of the 2007 Law of Historical Memory to focusing on a specific historical memory project that I had several opportunities to interact with during my study abroad. This project revolved around the exhumation and commemoration of the victims of Franco’s army at the village of La Sauceda in Andalucía, near the boarder of the provinces Cádiz and Málaga. While the inefficiencies of the Law of Historical Memory were still relevant to my discussion of the project at La Sauceda, I decided to more broadly evaluate all of the challenges that the project faced, along with the reasons for which the project has been successful .

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Troubleshooting

Start of the third week and back to performing CAT ELISAs. I am constantly reminded of how important getting the details right is to successful experimentation. Some of these details that I have failed to remember or perfect in past and current CAT ELISAs are: making sure the protease inhibitor tablets have completely dissolved in the lysis buffer before adding the lysis buffer to the plates, inverting the CAT Enzyme Working Dilution tube multiple times and pipetting the solution up and down to try to ensure a uniform distribution of CAT Enzyme within throughout the solution, making sure the cold centrifuge is set at 4˚ Celsius, checking the multi-channel pipette to ensure that each pipette tip has actually taken up solution, loading the sample trays in the CAT ELISA plate in the correct orientation so the plate reader will read them, and famously (from my first attempt at a CAT ELISA), not using working dilutions as washing buffers. These are only small parts of the CAT ELISA procedure, but remembering and performing them makes all the difference in the world.

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