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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).
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