Peddie EXP 2017

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 blogpost is a little late considering my last day was this past Friday, but I have been on the road since then and have not had the time or internet to write up my final entry to the blog and submit it. This last week was very, very busy in a good way.  The week started off with a refreshing trip blue berry picking with the lab, this was definitely a highlight of my time at Duke.  The entire lab dropped everything and from 9am to 11am Monday morning we just picked fresh, delicious blueberries.  It was kind of refreshing spending time with the entire lab outside of the lab. After that, however, I had to grind out the last of my projects as well as my presentation.  That week I conducted two behavioral trials with mice, prepared another whole round of DNA samples, finalized a protocol for the lab, investigated a faulty centrifuge, sorted and organized the HuOR library, created my EXP night poster, and then transposed that poster into a PowerPoint.  Suffice to say a lo

To start week 4 I was introduced to Matias, another graduate student in the lab. Since Hannah, my graduate student, was going to be away at a conference, I was going to be working with Matias for the next few weeks. On Monday he showed me the project he was working on and where all his materials were kept. In the afternoon, we went down to the fish facility and he showed me where all his fish were and gave me two tanks that would be mine. Today we discussed some genotyping which I was going to do for him. He told me it was some complexing genotyping and would require longer than a week but he thought I was ready for the challenge. There was not enough time to start the process today so we decided it would be better for me to start tomorrow. One of my tanks On Tuesday, I went down to the fish facility and began fin clipping. Each tank had around 50 fish so my plan was to fin clip one tank today and the other on Wednesday. The fish I was working with had just been born a couple of

With one more week to go, I have been trying to finish up my project and prepare for my presentation both in school and in the lab. I have officially finished analyzing the recorded calls and have added my data to the file already in the database. At this point, we have over 10,000 data points which will be used in determining the acoustic boundaries of the USVs and thus how maternal mice are able to categorize and understand the pup calls despite alterations. The next step is to graph this information and compare it with the recorded behavior and neural activity of the mothers when exposed to the USVs. The tones, frequency, speed etc. per age will be used to determine a general structure for an average pup call which will then be played back to a mother with varying alterations. By process of elimination, they will be able to understand what the mother is hearing and how she is categorizing the sounds she hears as calls. They have been procrastinating the analysis of these calls

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.  These were put back in the incubator.  

Week 4 and 5 were mainly focused on the same thing, transforming bacteria.  Since last week, the Y2F plasmid was not cut properly, yielding many bands of different sizes, rather than a band around 6k and one at 350 base pairs long.  We first double checked the plasmid map to make sure that the enzymes that we used were in fact the correct ones.  They were, so we contacted the company about the improper cuts.  They did their own tests and found that the enzymes were manufactured improperly.  They remade the construct and sent it back.  This time, the proper bands appeared.  We then had to send these bands out for sequencing to make sure that the inserts were in fact the mutated form of the Y2F insert, since the mutant and wt versions are the same length.  The image below shows how sequencing is done. The sequencing for the CAR19 with a Y2F insert came back with the results that we expected, the bands matched the mutated version exactly.  From here, we needed a larger colony of

               Like I expected, the last week in my lab was pretty quiet. Because there were a lot of psychology conferences going on around at that time in the summer, a lot of people were not in the lab. Also, because it was my last week in the lab, last week was the last time that I was going to see some of the members of the lab. So, again, I had to say goodbye to another one of the members of the lab that I had gotten to know very well, Yang. What’s great about being in a professional setting is getting to hear the experiences of the well-accomplished people around. Yang recently got the title of a Postdoc and during my time in the lab, I got to hear a lot about her journey from her undergrad years in China to her postdoc years in Cornell. Members like Yang really have inspired me to pursue what I’m passionate about. Throughout her years, she has always been really interested in learning about psychology in children. So, she has gained all of her degrees in that area and now,