Peddie EXP 2017

This past week was my final week at the Seale Lab. It started quite slowly, but the last two days were perhaps the busiest days I had during my time at the lab. So what happened? Monday was quite a slow day. I arrived at the lab at 8:30 AM as usual, and began working on my poster. I continued working until 10:30, when I realized my supervisor still wasn't at the lab. In fact, almost no one was at the lab except me and two undergraduates. I was a bit confused, but I continued working on my poster. Finally, at 1:00, I left the lab. I later got an email from my supervisor saying that I didn't need to be there that day.  On Tuesday, I had a good amount of work to do. I transfected the virus-infected HCT116 cells with p53 and empty pcDNA plasmids. Besides that, I genotyped 20 more mice, and worked on my poster. Wednesday was much like Monday, in that it was a slow day. I worked on my poster once again, and genotyped another 15 or so mice. Additionally, I spent about an hour pre...

This week was a rather short one, as I had Friday off, due to Rachel, my supervisor, not being at the lab. Overall, it was a slower-paced week, which was a welcome reprieve from the extremely packed previous two weeks. I spent a good amount of time working on my poster, but I also did some lab work as well. The first task I had was to genotype even more mice to see if any one of those mice were triple knockout for Prdm16, p53, and Casp8 . At first, I thought I would be let down once again, as in the 60 mice I had previously genotyped, I had not found a triple knockout. However, I was pleasantly surprised to see that not one, not two, but three of the mice I genotyped were triple-knockout mice. This was a great success, as these are the first triple knockouts we've seen in the seven weeks I've been here. However, all of these mice were male, meaning that we couldn't just breed these triple knockouts together to produce more triple knockouts. Nevertheless, it still was a go...

This past week was one of both successes and failures. On the one hand, my Co-immunoprecipitation  experiment went well, and we were able to establish a link between Prdm16 and p53. On the other, we had a spate of bad luck concerning transformation and plasmids. The week started off fairly normally. I spent that Monday preparing and running my protein gel. I found out rather quickly that protein gels were nothing like agarose-based gels; protein gels were much more difficult to load precisely, and the gel had to be handled with the utmost care. While the gel was running, I busy preparing a presentation I would give during lab meeting the next day, and I was having quite a bit of trouble. I didn't quite know what to say, and I had no results I could show in time for lab meeting. Finally, I settled on explaining what I was doing, why I was doing it, and expected results (almost like the spring term EXP poster, in a way). Thankfully, I was not going to be the sole presenter, as tw...

So these past two weeks were extremely busy, and quite eventful as well. Most of the week was spent trying to transform DH5a competent cells with specific Prdm16 plasmid we made using Gibson Assembly. We tried increasing the concentration of plasmid, using new Gibson Assembly master mix, and even tried to run a positive control to make sure everything was working. All of our attempts failed, indicating either the cells or the master mix was the problem. So I transformed pure plasmid into the DH5a cells to see if these would transform, and they did...sort of. There were indeed bacterial colonies, but our transformation efficiency was 100x lower than what it should have been. This obviously was a problem, so we ordered new, ultra-competent bacteria to see if we could at least transform those. And indeed, our Gibson Assembly worked. All total, we spent about one-and-a-half weeks just trying to get the transformations to work. However, that was not the only project we did. In addition to...

Note: Weeks 1, 2, and 3 all blend together, and so I decided to make a blog post for all three. Week 2 was my actual start at the lab, and Week 3 was a shorter week, due to the Fourth of July Holiday. So far, my lab experience has been extremely interesting and fun, if not a bit surprising. I came into this lab thinking I was going to be working with brown fat, as that is the main focus of the lab, so I was quite surprised when I found myself working with intestines instead. Before I came to the lab, my supervisor had found that in mice, whole body deletion of Prdm16 , which is required for brown fat development and maintenance, led to greatly reduced intestinal length, killing the mice within seven days of Prdm16  knockout.  This phenomenon had not been previously observed, and so the project I'm working on is an entirely brand new area of research. My first task was to genotype mice and find a particular mouse with Rosa26CreER,  an on-off switch that can be activate...