Peddie EXP 2017

This past week was my last in the lab.  In the middle of last week, I continued to work on immunofluorescent staining.  This took a couple days to finish because the antibodies need to be left on the staining slide overnight.  I added 2 more antibodies: DAPI which stains for nuclei, and another general stem cell marker called ­­­­CD271.  When I originally observed the stem cells under the confocal microscope, I did not see what I was expecting to; instead, I saw blurry colors that were not vibrant at all.  This made me quite nervous because this is essentially what I had been working towards for the better part of six and a half weeks.  After looking at the stained cells under a regular microscope, we discovered that in each of the 8 slides, the cells had grown primarily around the outside of the slides and sparsely in the middle region.  With this new knowledge, plus the help of Dr. Du, we were able to take some great pictures, some of which are show...

After having injected the gRNA constructs I made with the cas9 to see if I could successfully knock out my target genes, I've mainly been focusing on helping out with other projects around the lab, mainly with Mercedes' project in synthesizing some other constructs. In addition, Collin has been instructing me on how to actually figure out how to pick genes to target in various metabolic pathways, and how to make up the gRNA construct on the computer. Since my gRNAs are for the murine (mouse) model, he has assigned me to creating one for a human model (which, when synthesized, will go into humanized mice , not actual humans).  This mostly involves research into the genes and metabolic pathways of interest and figuring out which exons are most important, and through using a few fancy programs on the lab computer, how to alter them in a way that can generate a frame shift mutation to knockout the gene. Although the computer work has still been interesting and no doubt important,...

      These eventful two weeks have proven to be pivotal; I have taken over yet another project. On top of the Anopheles funestus optimization, the modelling of a gene drive system, and the cloning of Homing Endonuclease Genes (HEGs), I have been directed to create a control procedure for creating variants of the AGAP007280 HEG.       In Week 3, I was assigned the cloning of a specific version of the AGAP07280 (7280 for short) gene. This was the 7280(2) version. Once I had cloned the gene through a series of transformations and bacterial plasmid extractions through mini-prep and maxi-prep techniques, I was shown many different variants and modifications of the gene. Some of the variants included a change in the HEG itself, others included MEGATAL endings, thus increasing the length of the sgRNA recognition site, and the most extreme modifications included a complete change of endonuclease enzyme within the HEG. Regardless of the change...

On Monday, I walked into the lab at 10 AM to find it completely empty. Turns out there's a flu going around in the lab. Luckily, I am intact from the disease. Unfortunately Ram and Mark were both sick from the flu so I'm left alone for the rest of the week. However, Mark and I still communicate through text and he ask me to grow several MoS2 batches through the week. There have been several problems that I ran into during those growth. First, there were leaks within the sulfur ventilation system that made the room smell like sulfur at one point. Second, when I was growing WS2, the flakes were really small with a low density. I'm not sure if there's a problem with the growth promoter or my method is malfunctioning. Before I had the chance to find out, I got switched back to growing MoS2. It seemed my series of unfortunate events didn't end with WS2, my MoS2 flakes didn't grow the first 2 times. People say three time's a charm and indeed it was, I found a pro...

Me and Deja in front of the chapel My last week in the lab seemed rather long, but it was a memorable one. We changed media on Monday, as usual. and did some behaviour testing. Tuesday was the same, except my lab threw me a little going away party. The conference room table was filled with the most Chinese food I have ever seen and they gave me a gift at the end. After lunch, we checked the status of our cells to make sure we would have enough to inject on Thursday. As we expected, our cells were growing very well and they were ready to be extracted and injected- we had to wait until Thursday because Yul was very busy helping people out in other labs. On Wednesday we sacrificed 10 more female mice just in case we did not have enough cells for the injections of group 3 and 4. Like before, we extracted their bone marrow and plated the cells. Thursday morning was pretty slow and uneventful. In the afternoon, Yul prepared male and female cells for injection. The male and female BMSCs...

Week 3 did not have much going on to start off the week because we received a four day weekend for July 4th. I spent Saturday-Tuesday in Florida with some friends from school before returning to Philly on Wednesday. In Florida I went to the beach, hung out at the pool, and ate lots of good food.  Spent July 4th weekend in Clearwater, Florida When I returned to the lab on Wednesday, there was a new undergrad who joined the lab. I was put in charge of getting him all setup. I introduced myself to him and he told me his name was Jack. I found an empty desk for him and he put all his stuff down. Next, I showed him around the lab and introduced him to all the members. The rest of the morning I spent teaching him basic techniques for the lab like pipetting and identifying/sorting embryos. Ben, a graduate student, took Jack in the afternoon and introduced him to the more complex techniques and helped him continue practice the basic techniques I taught him. Although I had only bee...

Just as the fifth week at the Lee Lab was about to begin, I received an email from my P.I. asking for me to prepare a report of some sort that summarized my work in the lab so far. As I already have a presentation file compiling all of my data as well as the parameters of my experiments on hand, I decided to use that as the starting point for my report. After organizing the data in chronological order of what I did during the past four weeks, I also added parameters of the experiments I conducted as well as observations that I noticed while performing the procedures. After I finished up organizing my data, I proceeded to share it with Jyo Lyn, my grad student, as I asked her to look over it before I shared it with my P.I. After she looked over my work, since the presentation file I shared with her did total up to 93 slides, she said that it was way too long. Therefore, she suggested another way for me to organize the data, by grouping them in a different way instead. After I re-organi...