Peddie EXP 2017

Week4:                 PCR, PCR, PCR, this week is just an endless loop of PCR. One big difference that I noticed between what I did in AP Chemistry or AP Biology and what I do in real-life research is that things are much more unpredictable in real life. In the labs made for those AP courses, there are standard protocols, standard instruments, thousands and thousands of other students and teachers had done these labs before, so what we are essentially doing is practicing our lab skill in a path that is already explored. When we do those experiments, we know they will work because that’s how they are supposed to behave. Things are much wilder and much more interesting in the real research field. First of all, you are doing what has never been done before or rarely has been done before by other people. You need to come up with your own protocol, and you need to read a lot of research papers to confirm the liabi...

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...

      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...

This is my first blog post in two weeks as both weeks have been shortened by the fourth of July and my grad student Joe’s mini vacation to New York City.  That said despite spending less time in the lab than previous weeks, I still managed to accomplish a great deal of work. I only worked 3 out of 5 business days during the week of the fourth of July.  After celebrating our nation’s independence over the four day weekend, I went back to work on Wednesday, picking back up where we (Kennadi and I) left off.  Using a QIAprep Spin Miniprep Kit, we isolated the DNA from the bacteria cultures that we grew in LB broth the previous week.  Additionally, this day marked the end of Khin’s (a graduate student) time in the Bouchard Laboratory.  We celebrated her many hardworking years in the lab as we welcomed a new transfer student who will be spending the next 10 weeks in the lab.  It seems like every week someone is either leaving or joining the lab giving me the ...

My fourth week at my lab got off to a busy start on Monday! I jumped right in with my mini-project given to me just last Friday, which took me the better part of a long (yet anticipatory) day. I'll take this moment to backtrack a bit and provide some information on my experiment. I am comparing the LD column and the autoMACS bead selection to determine which will result in maximized cell enrichment, as well as which will allow for (n oncontaminated) cell culture; I am also going to determine what volume of selection beads to use to filter out  as many unnecessary cells as possible. If I were to simplify the description completely, the LD column is basically a magnetic field. The user adds magnetic "beads" to the sample and these beads adhere to the cells that have a certain marker (e.g. CD45+ cells); the magnetism of the LD column basically holds the magnetic beads (and thus the attached wanted cells) while letting all other unmarked, non-magnetic cells filter through....

Finally, after the arduous process of preforming a Maxiprep, I’ve successfully created my gRNA construct. Amazing, the process of 6 weeks of straight work, all for two little tubes of 300 microliters of what looks identical to water. The constructs were injected into two mice down in the mouse lab, which apparently I’m not even authorized to set foot in, to my disappointment. Dr. Pankowicz and Collin both assured me, though, that the injection was successful and within a couple weeks, we should have some mouse livers to harvest, and truly see if the construct was successful and if the genes of interest were knocked out.  Maxiprep materials Some appetizing cell debris after the DNA has been removed (step 5 of the maxiprep) The Maxiprep itself was especially annoying, only because I messed up the first step for close to a week straight. A Maxiprep describes a process where you can amplify and get large volumes of plasmid DNA in for processes like injections, when a Minipre...

Bowling with friends On the first day of week 3, Shawn and I changed media and did von Frey testing on the male mice. As usual, their pain threshold was higher than the female's and they were less fidgety. The following day, we sacrificed 10 more mice: 5 males and 5 females. We plated the bone marrow cells as we typically do and continued to change the media for all of the batches of cells. Shawn removing bone marrow using a syringe and tweezer The hood where we change media and plate cells  Additional image of female BMSCs plated on 6/14/17 Image taken on 6/30/17 of female BMSCs plated on 6/14/17 On the Wednesday of week 3, we made new media because we were running low. We put two antibiotics, penicillin and streptomycin, and fetal bovine serum (FBS) in the media. Though I knew why we put the antibiotics in the media, I did not know why we put FBS in. As it turns out, the FBS provides proteins that are necessa...

Week three and four have been very eventful and promising. In the beginning of the third week, I had my mouse lab orientation, which now means that I have granted access to the mouse room by myself! Although it took longer than I expected for me to get this, I am excited that I have my own independence now to visit the mouse room. Elizabeth sent the lung samples that we collected from the dissections the previous week to the Duke CORE facility to be embedded in paraffin. It takes at least a week to get the sample back so in the mean time we have been working on the ex vivo project involving the bone marrow derived macrophages. Since Sarcoidosis is a chronic inflammatory disease and its pathogenesis is still unknown, we are only using the macrophages to see the granuloma structure under a microscope, if there are increased expression of specific genes during the disease, and how different concentrations of carbon nanotubes affect granuloma growth. Our main gene of focus that week...

My fourth week at the Khalizov lab has concluded. While I only have two weeks left in the lab, the experiment is at a standstill since the mass spectrometry is still malfunctioning. Matt and Dr. Khalizov are still working on the mass spectrometry. Unfortunately, hopes are low currently. While John and I are not allowed to fix the mass spectrometry due to its technical nature, Dr. Khalizov tried to explain the problem to us on Monday since we have limited work without the machine working. Apparently, the triangular turbine in the mass spectrometry is malfunctioning because of some resistors arranged in a wrong way. This prevents reagent ions to run the mass spectrometry. Another potential problem is contamination of the mass spectrometry, which may be giving some odd values. Dr. Khalizov is planning to call the technician from the company that made the mass spectrometry. While it does not look promising at the moment, I hope John and I can perform some experi...