Week Nine

I started my final Western Blot on Friday and let it run while I was at my lunch lecture. However, when I came back all of the members of the Padiath lab seemed very concerned with the progress of my gel - the proteins had stopped moving, even the ladder was no longer moving. The lab tech asked me if I had heated the protein samples before loading them, and I hadn't - it was something I forgot to write in the notes I was following so it completely slipped my mind. Without heating the proteins, they do not become denatured and even if they had continued to run down the gel during the electrophoresis stage, the locations they ended up on the gel would have been inaccurate because the size of a protein folded into tertiary or quaternary structure is quite different from the length of its unfolded primary structure as an amino acid chain. The reason the proteins were not moving was because our apparatus and power sources have been not been working as they should, and sometimes stop the electrophoresis from progressing. Since I already missed an important step, it was actually helpful that the equipment malfunctioned because otherwise I may have gone through the rest of the Western Blot and only realized I had missed a step once I was analyzing the results. 

Figure 1. Simplified drawings comparing folded and denatured proteins (RSC).

The graduate student, lab technician and I are now trying to insert a gene into the plasmid DNA we had replicated through bacterial transformation a few weeks ago - a process called cloning. Plasmid DNA can be cut in specific spots with restriction enzymes, and closed to create their circular shape again once the insert has ligated to the cut DNA. As PCR is a common technique in molecular biology, various polymerase chain reactions are carried out throughout the procedure, requiring very specific primers for each step.

Figure 2. Outline of the plasmid cloning process (Addgene).

For our final lunch lecture, one of the current co-directors of the Center for Neuroscience's Graduate Program talked to us about their program and graduate school in general. He discussed many skills and desires that someone needs to be successful in graduate school, and it assured me that I am still on track to pursue a Ph.D. The Center for Neuroscience's graduate program prides themselves of sponsoring scientists from all different kinds of backgrounds, so if I decided that I wanted to apply to their graduate program, it would still be an option for me as a chemistry major, which is wonderful news.

Figure 3. Dopamine, a chemical crucial to proper function and response of the reward system of the brain.

I had to write an abstract for my project this week, and will give a presentation on my work next week. Writing the abstract allowed me to collect my thoughts and gain a better understanding of the relevance of my project to the disorder I am studying. My abstract will serve as a summary of my time in this lab and all I have achieved as I have been here, while my presentation will allow me to share my work with the other fellows and the directors of my program, as well as any other guests that attend, including my mentor and possibly one of the Padiath lab members. I will be collecting data for my presentation right up until it is due, as well as some data for my project that I will only be able to collect after my presentation is due, but is important for the future of the project.

Wednesday was a fun day because we had a birthday party for one of the women who works on my floor as a lab tech and is also a graduate student in our department. Some of the people on our floor bought her cakes from Oakmont Bakery that we were all encouraged to eat, which was a nice break during the day. She is incredibly friendly and talkative, and I am happy to have been able to make a connection with her and other people who work on our floor, even though they do not all work in the Padiath lab.


References:

Addgene. n.d. Plasmid Cloning by PCR. <https://www.addgene.org/plasmid-protocols/pcr-cloning/>.

[RSC]. n.d. Enzymes. <http://www.rsc.org/learn-chemistry/resources/chemistry-in-your-cupboard/vanish/8>.