Doug Reinisch Week 2

My second week in Philadelphia was much more compacted in terms of both work as well as other “obligations” while in the city.  That said, although I had less free time I feel that I benefited more from learning more about the research and the city itself than I did in my first week.

On Monday, we continued our mutagenesis project as we began the day by conducting gel electrophoresis.  Kennadi and I worked together to make an agarose gel to run the mutant HBx samples on.  The entire gel electrophoresis process itself was familiar to me, thanks to ample practice doing so in biotechnology.  After the gel was finished, we stained the gel with Ethidium Bromide (EtBr) in order to be able to view the DNA bands under UV light.  After determining that all of my three samples and 1 of Kennadi’s were the only successful ones after viewing the gel, we conducted PCR on all the failed samples (Joe’s 2 samples and 2 of Kennadi’s).  The goal of this was to raise the Tm (annealing) temperature of the PCR reaction in hopes that there would not be any nonspecific binding this time.  The beauty of conducting real science is that if you fail one time, it doesn’t mean that the theory is flawed, it may just be one step in the sequence that needs tweaking.

Our DNA in motion

Tuesday was very similar to the previous day as I helped Kennadi run her failed PCR samples as well as both of Joe’s in hopes of different results.  We ran into the same issue as before on this gel as we had a significant amount of nonspecific binding occur.  Therefore, Joe suggested that we make and run a third gel, but this time cut out the DNA of interest under very low UV light in hopes of not damaging the plasmids.  Our HBx plasmids were located between 6,000 and 7,000 base pairs, while we had nonspecific binding both above and below this range.  After isolating this part of the DNA gel, we performed DNA gel purification to remove our genetic material from the agarose gel.  We made the assumption that this band would work just as good as the successful PCR trials in later stages of our mutagenesis project.

Our failed gel from Tuesday (the DNA ladder is on the left)

Wednesday was a much lighter day for me.  I did not do any actual science the entire morning before lunch.  Despite there being a lesser load of work, I did spend several hours in the morning learning more about the field of Hepatitis B Virus (HBV) and liver cancer.  Joe went into much more depth about the theory behind not only the project Kennadi and I are helping him with, but also the entire field of the HBV-HCC (hepatocellular carcinoma) pathway and the role HBx plays in it.  After taking a lunch break, we ligated some of the remaining PCR product into bacteria culture and grew them on agar plates we made the previous week.  To do this we carefully heat shocked the bacteria which opened pores in the membrane of the bacteria to take up the DNA plasmids, but not too long that the bacteria would die.  The reason we had to wait until the afternoon to do this is that once complete, the bacteria need 16-18 hours to grow in the incubator, thus we wanted them to be finished at the start of the day on Thursday, not in the middle of the night or else they would overgrow the individual colonies.

Joe's "artwork"

As stated previously, the bacteria finished growing right when we start work in the lab in the morning (approximately 9:00).  Therefore, we immediately removed the cultures from the incubator and determined which of the agar plates were successful.  The plates with many individual colonies as expected we used parafilm to prepare them for long-term storage.  The unsuccessful trials (mostly the ones that failed the gel electrophoresis earlier in the week), we redid the ligation and regrew them on different agar gels.  Again, we had to wait until later in the afternoon to complete this to avoid having the bacteria outgrow the individual colonies.

The last day of the week was by far the most hectic that I have had in the laboratory thus far.  Fortunately, I did not have to come into the lab until 10:00, that said once I arrived Joe had me running up and down the hallways in the building with him, rounding people up to come to the presentation at 10:05.  Khin, a Ph.D grad student, was giving the presentation, defending her thesis and putting an end to her research in the Bouchard Lab.  Khin discussed her findings about HBxIP and people from all parts of the biochemistry department, as well as former members of the department were in the audience.  Following the presentation I had to race off to the subway station at Market Street in order to ride the Market-Frankford Line (MFL) over to University City to meet with Dr. Peretz, Mr. Sham, and 7 other EXPers for lunch.  We enjoyed a delicious meal at the White Dog Café, as everyone shared their unique experiences thus far with one another.  After the long meal, Mr. Sham and I rode the MFL back to center city to visit my lab.  We sat down with Dr. Bouchard, my PI, in his private office and the three of us discussed the goals of my research.  I then proceeded to give Mr. Sham a self-guided tour of the lab, met my colleague Kennadi Cook and he took a few pictures of me “hard” at work (the pictures were staged).  Unfortunately, Joe was at lunch and Mr. Sham was not able to meet and greet him.  After Mr. Sham left and Joe came back I worked alongside him (Kennadi went to a presentation for her program) and we transferred 2 individual colonies from each successful agar plate into a test tube with LB broth to grow the bacteria in.  After placing them in the incubator I said goodbye to Joe and the rest of the lab as I left for a long 4 day weekend.

The group at White Dog Cafe

Overall I enjoyed my second week much more than my first.  I am most proud of myself for taking the subway by myself after never previously taking a Philadelphia subway and correctly guiding myself to lunch.