Skip to main content

The Last Week- Eric Veltri

This week started off great, I was finally able to get results from my qPCRs of the lncRNA, PACER, in addition to performing a successful sub cloning experiment. On Monday, I worked on preparing my one E. Coli colony for sequencing to confirm the success of the transformation. Once the results came back later on that week from the sequencer, it was official that the plasmid from the bacteria contained the COX-2 gene (the goal of the sub cloning). The purpose of making these plasmids is so that Liz would be able to have more control of COX-2 expression in her cholesterol effluent experiments in human and mouse macrophages. 

 In addition to getting the subcloning results, I ran two qPCRs to compare PACER’s expression when treated with different ligands and to compare PACER’s expression in mice to that of humans. My conclusion is that the PACER gene in mice is induced similarly to that in humans despite the sequence and length being significantly different. These qPCRs helped Liz’s project by answering one of her many questions about how PACER is different and similar between two species. She is hoping to design further experiments to see what other aspects differentiate the mouse and human lncRNA.

 Since it was my finally week and I got my results earlier than expercted, I spent the remainder of the week helping Liz with her mice and other post docs with their projects. I got to help Damien with Western Blots, a process similar to gel electrophoresis, but for proteins. The Western Blot has so many steps to it, that it’s very easy to mess up, but our Western was a success. The coolest experiment I helped with this week was watching a live surgery of placing a mouse under anesthesia, and then using a intervivo microscope to puncture an arteriole with a laser. To prep for the experiment, I had to reuse my immune histology technique from my microscopy and tag platelets with fluorescent antibodies. With the software associated with the microscope, the post doc and I were able to see how the platelet aggregation in wild type and COX-2 KO mice was different. Since this was live, I got to see the blood flow in the artery in the microscope and I was able to use the fluorescent light to see the platelets arrive and leave the site of injury.

 Below are pictures that I decided to take of techniques that were part of the qPCR and subcloning experiment, in addition to other photos that I chose to take of the lab.

 This is the large well plate for the qPCR. (I definitely messed up a few times, loosing where I was with either the DNA or the master mix)
 "Air drying" the pellets of DNA.
nanodropper

 This is a graph from the nanodropper. In green is the concentration of the DNA.
 This is an opened nanodropper ( a smaller mass spectrometer). I used this to get the quantity of the DNA that I extracted (in nanograms/microliter).
Labels:

Comments

Post a Comment

Popular posts from this blog

Evan Bradley, Week 3 at the Missouri Orthopaedic Institute

As mentioned in my previous blog, I have been awaiting ligament, menisci, and cartilage tissue from a canine or human knee joint for (interleukin) IL-1B tissue culture. IL-1B is an inflammatory cytokine that has been proven to increase rates of tissue degeneration and osteoarthritis development in the Thompson Lab. Dr. Stoker wants me to experiment with different types of knee tissues in a co-culture with varying levels of this cytokine to determine its effects on the entire knee joint. This co-culture uses an insert permeable to the media to separate the two tissue samples from physical contact, while allowing them to share the same media. This creates an extremely accurate model for knee tissues in their native environment due to their exposure to the same synovial fluid in the joint. This model would then be treated with the IL-1B and cultured for 21 days. During these 21 days, the media would be collected every three days for biomarker evaluation at the end of the stu...
Post a Comment
Read more

Kylie Heering, Week 2 at the Goldstein Lab

We started off our week with a congratulatory acai bowl trip to celebrate Preston’s acceptance into a training grant program. Acai bowls in California top Playa Bowls (no question about it). From what I can tell, its a pretty huge honor to be recognized by this grant, but he’s really humble about it. On Monday, Preston and I decided that testing antibodies that have never been tested on prostate epithelial cells before would be a good objective for my first Western blot on my own. We needed to probe for ASCT2, a glutamine transporter, and GLS in order to determine if their corresponding antibodies are functional. Antibodies are crucial for Western blots because they bind to the protein of interest (POI), allowing for us to qualify its expression after imaging. As such, Preston wanted to make sure they worked by probing for ASCT2 and GLS on three different cell lines. Cell lines are commercially purchased human cells that have been immortalized (modified to grow indefinitely) by telome...
Post a Comment
Read more

Daniel Cheng, In My Own Room

I had no idea that Pennsylvania is this wide. Within the first hour, my train had reached Philly. But to Pittsburgh, it took another seven. Even before I stepped foot into the Search-Based Planning Lab, I was waylaid by some anxious news. The PhD student assigned to be my mentor, Dhruv, texted me that he, Dr. Likhachev, and most of the lab would be out of town for the entire week. So that was that. Fortunately, it was my first week, the week to be spent learning new material, and Dhruv provided me with plenty to digest. I already had ROS (Robot Operating System) installed, so I looked towards the tutorials that ROS provided. I copied commands into my Linux laptop's terminal to run ROS features. I learned the basic structure of ROS: packages, services and clients, publishers and subscribers, messages, nodes, and topics (which nodes communicate messages over). There was one simple yet interesting program I came across in the tutorials called turtlesim, for which using only 2 comm...
3 comments
Read more