Peddie EXP 2017

Hey guys! I have decided to combine these three weeks in one blog post because I really only go into the lab space two or three times per week, with the rest of my work being done at home. So the reporting on actual lab events is a little scarce. These past three weeks have definitely been interesting. After the big grant was completed, there has definitely been a major shift in the lab’s environment, a lot less tense and overall a better energy. Week 4 started off with a lab meeting involving me, my post-doc Kayla, another student Nick G, and our lab’s PI Nick T, all had a meeting about this metabolomics project. I have been preparing for this meeting essentially for the past three weeks and was ready to discuss. Yet within the first 5 minutes of the meeting, it was clear that the goal of this project was actually changing, which sort of threw me for a loop. But in our project, which is still in its conceptual phase, things are ever-changing. I found out about this document for a pro...

My time at Hahnemann Hospital at Drexel University just came to an end this past Friday, but not without making some great memories in my final two weeks working in the Bouchard Laboratory.  Both inside the lab and out, I tried to make the most out of my limited time left in Philadelphia this summer. The view of Hahnemann Hospital at Drexel Medicine from 15th Street. My sixth week started off working on the new mutant sample (HBx 50-131) that we had begun work on the previous week.   Kennadi and I performed a QiaPrep MiniPrep Spin Kit on the bacteria cells with our mutant DNA.  Unfortunately when we used the Nanodrop One to determine our concentration and cleanliness of our plasmids we found that something either went wrong with the transformation or the MiniPrep.  Afterwards, we continued to track the growth of our HepG2 cells on the collagen plates until it was time for the weekly lab meeting.  Ron gave the presentation at the lab meeting about...

For this week we really focused on running our Mass Spec machine in order to get a bulk of our filtered water samples processed so that we could move on to running other data that had been collected in the field. The figures above show what our Mass Spec machine looked like. It really consists of 2 components. One, the "EA" or Elemental Analyser, and the second part, the actual Mass Spec machine. These two components work together to give us the data that is needed To run these machines, however, tons of preparation and tiny details must fall into place.  First, the EA needs to be brought up to temperature to ensure that each capsule we place will combust. Second, we must pump oxygen for a multitude of reasons in order to keep the EA functioning. And third, helium must be running through the system to ensure that the combusted CO2 and N2 reach the Mass Spec machine and get processed.  Other than these details, each furnace of the EA must be packed with sp...

         Although there were only three days to Week 6, these three days will determine the success of my generated 7280 HEG variants.         On the day that everyone returned from the lab conference in Crete (coincidentally the same day that I returned from a Greece trip of my own), the song, "The Eye of the Tiger", was blaring throughout the lab.         Dr. Drew Hammond was very excited shouted, "Injection day *plural expletive*! It's the moment of truth to see if your plasmids work within the mosquito genome! Better hope they home!"         I laugh not only because of the unique British expletive, but also because it had been about the third repeat of the song. Luckily, the song did not play for the entire day; they also played "The Final Countdown" by Europe and "Don't Stop Believin" by Journey (so pretty much the same song all day lon...

Week 6 was my final week! The time went by faster than I ever could have imagined - six weeks seemed a daunting stretch of time at first, but I realized how short it really was as I started my sixth week at JHU. I wrote up and finished my bone marrow monocyte isolation protocol over the weekend and began my final week by going over the protocol with Snow. I got a clearer sense of what we would be doing, why we were doing each step, and what the overall purpose of the experiment; she answered all of my lingering questions and provided helpful tips for me to keep in mind in anticipation of and in preparation for the experiment. For clarification purposes, my new protocol was called "Bone marrow monocyte enrichment using the Miltenyi bone marrow monocyte isolation kit" (Miltenyi is the name of the company that makes the enrichment/isolation kits). We wanted to look at how many monocytes could be obtained from the bone marrow, as well as test out the aforementioned Miltenyi k...

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...

This week was all about making viruses.  I needed to make a Virus using the CAR19 + Y2F plasmid as well as 3 other viruses.  The process of making viruses is initially very complicated, but toward the end of the process gets much clearer and simpler.  There are a ton of reagents that need to be mixed with the plasmid DNA and the 293 cells.  This cell line is used because it is one of the easiest to transduce.  The process of making the Lente Virus began by mixing the plasmid DNA, 293 cells, and reagents that allowed for the plasmid to more easily transduce the cells.  These cells were then put in an incubated flask at 37 degrees overnight with 60mL of media. The next day, the media had been almost completely drained by the cells and was now filled with the desired viruses.  We removed this media, collecting it in 2 50mL conical tubes and added a fresh 60mL of media to the flasks so that they could continue producing more virus.  ...

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...

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...

It was pretty sentimental thinking about leaving Duke after this week. I learned how to do so much in the lab and I enjoyed exploring all the great places Durham has to offer. This week I had a little more free time during the day since there wasn’t much left for me to do so I took the opportunity to finish my EXP poster knowing that I would not want to do it when I got home. I even showed it to the other members of the lab when I was finished and they were amazed. One couldn’t believe that I was still a high school student. On Monday, Elizabeth and I set up our plates for the vitamin D experiment. For the past 6 days, the bone marrow derived macrophages were sitting in the incubator in order to stimulate growth. Before we set up the plates on Monday, I looked at them under the microscope and they looked great! The standard concentration of cells in a plate is 1x10 -6 cells per mL. If the cells aren’t at that concentration, then you have to dilute them with PBS, which is what I d...

After weeks and weeks of trying and failing, I finally hit the nail on the head. Yayy! I figured out how to display the number of pixels in the census transform. Overcome with excitement, I tested it out on a few more picture sets, and it worked without a problem. I showed Dr. Taylor and he was very pleased with my work (whew!). He then explained to me the next step in my task. It is very similar to what I have done, but it just applies the information differently. My task is to make a histogram that shows how many times an 8 bit pattern from a census transform occurs. Once the 3x3 pixel square undergoes a census transform, it become a binary, meaning it is solely zeros and ones. However, in a sequence of these zeroes and ones you can add them up to get one number ( i.e. 0 0 1 1 0 0 1 1 would equal 51. It sounds weird, right? How does that many zeroes and ones add up to 51?? Well binary is read from right to left, with the right most integer being 2 0 , then 2 1 , and so forth. You th...

My lab during an ice cream outing at Toscanini's :) This week, I proceeded to attempt converting a raw SEM image into a binary array. Although the code successfully segmented the images and calculated the centroid coordinate points, the order in which the coordinates of the centroids were listed in the data table was incorrect. Upon research, I realized that the root of the problem was the method in which MATLAB’s ‘centroid’ function saves the coordinate points of the centroids. This led to incorrect calculations of the state values, and thus an inaccurate binary array. To overcome this problem, I manually rearranged the coordinate points so that the y-coordinates would be in the correct order of descending posts from top to bottom (rows) and left to right (columns). As a result, the code produced a binary array with the correct numerical values, as well as a visual representation identical to the block copolymer pattern shown in the raw SEM image. ...