Doug Reinisch Week 5
My fifth week at the Bouchard Laboratory in Center City Philly was filled with both the end of one project and the beginning of a new one. I also had time to explore new parts of the city on Friday.
Monday started off the same as every other week with the grad students working on a rat students and a new member of the lab being introduced to all of us. Later that morning Joe taught Kennadi and I how to send the completed plasmids for our 7 mutant HBx proteins off for sequencing. It consisted mostly of diluting our plasmids with ddH2O and labeling of small tubes. After a mini lunch break Kennadi and I changed the growth media for our HepG2 cell plates (her's were contaminated so I gave her one of my 2 plates), because the cells were clumped together on them. To end the day we each made 500 mL of 1x PBS, which is used in cell work, from a stock solution using the autoclave machine. This PBS would be used by us later in the week.
The following day we used glycerol to make stock solutions of our samples to deep freeze in the -80 degree Celsius freeze. This was in anticipation of 100% success rate on our sequences that did not come in until the afternoon. When they finally did arrive, using a program on ncbi, we found that we did in fact have 100% success rate on our samples. The next part of this project is a transfection (mutant plasmid going into human hepatocytes) which in all likelihood will be completed mostly by Joe, hopefully before Kennadi and I leave the lab in the coming weeks. To end the day we each split our 10 mL HepG2 cell plates onto 2 new 10 mL 3x collagen plates.
On Wednesday we began with a mutagenesis reaction (PCR) for a new mutant protein. This time we were trying to create an HBx protein with only amino acids 50-131, making deletions at both the n and c terminal of the protein. As the thermal cycler did its job, Joe showed us how to do a trandfection with one of his other projects and we then were able to view both the mitochondria of the cell and the mutant HBx protein under a microscope, thanks to MitoTracker (stains the mitochondria deep red) and GFP (illuminates HBx protein green).
Monday started off the same as every other week with the grad students working on a rat students and a new member of the lab being introduced to all of us. Later that morning Joe taught Kennadi and I how to send the completed plasmids for our 7 mutant HBx proteins off for sequencing. It consisted mostly of diluting our plasmids with ddH2O and labeling of small tubes. After a mini lunch break Kennadi and I changed the growth media for our HepG2 cell plates (her's were contaminated so I gave her one of my 2 plates), because the cells were clumped together on them. To end the day we each made 500 mL of 1x PBS, which is used in cell work, from a stock solution using the autoclave machine. This PBS would be used by us later in the week.
1x PBS I made on my shelf
Joe's shelf in the deep freezer (-80).
An example of GFP and MitoTracker as seen under a microscope (from an earlier project of Joe's).
I finally got to sleep in for the first time during the work week on Thursday, because my only morning requirement was to meet with Dr. Bouchard at 11:00 to discuss what I have been doing in the lab as well as focusing on how to make my presentation both at the lab and at Peddie in the fall as successful as possible. With a commute of roughly 75 minutes, not having to get into the lab at 9:00 really makes a difference in the energy I have in the morning. In the afternoon we made and ran a DNA gel for our new protein and after the gel confirmed we had a successful PCR reaction from the previous day, we did a ligation (making protein circular again) and transformation (growing bacteria infused with our plasmids on an agar plate) for HBx 50-131. Unfortunately, very few colonies grew after the transformation and incubation.
Agar plate from previous study with lots of colonies (left) vs. Agar plate for HBx 50-131 (right)
My fifth week came to a close with a rather short day on Friday, giving me time to explore the city on my own. But first, I had to grow up my colonies from the agar plates in 5 mL of LB broth which contains ampicillin, the ampicillin resistant gene in our bacteria allows it to survive while killing off all other bacteria (assuming they don't have the gene). Joe incubated them over the weekend for us when he came in on Sunday. After this all we had to do was split our 10 mL plates onto 2 more 10 mL plates as well as a 6 well plates (cells left over were thrown out). These too were incubated at 37 degrees Celsius over the weekend. After this brief, yet meaningful day I took the time to visit the historical part of Philadelphia and I went to the new Philadelphia Museum of the American Revolution (opened in April). The museum of course focuses on the later half of the 18th century, but also does a nice job describing how the revolution lives on (abolition of slavery, women's suffrage, civil rights, etc.).
Overall it was another solid week in the lab, luckily filled with high success rates as my time in the lab starts to wind down. I am going to be working all next week and an unknown amount in my seventh week (depends on if I finish everything I'm doing or not) and then that's it for my summer EXPerience.
The 2 10 mL plates and a 6 well 3x collagen plates I split my HepG2 cells onto.
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