Week 9 and 10: Pufall Lab, University of Iowa

July 31st, 2014

John Christiansen ’15, Dimensions Fellow in Research

I have condensed week 9 and 10 into a single entry because week 9 was relatively uneventful and I was very busy in Week 10.

Week 9 was the last quiet week. The FACS sorted well had begun reaching significant cell density; some of the wells began to change from a light pink color to an orange, indicating an increase in cell density. However, it was still too early to move the living wells onto a new, live-cell only plate. On such a plate they would more easily be assayed for cell density, and the proper number of cells could be harvested for PCR of a segment of the BCL6 gene. From the size of the PCR product, we could determine which cells had the desired deletion.  This process requires the FACS sorted cells to grow to a significant density, as well as BCL6 gene segment primers of the PCR.

Miles and I also began to seriously consider being a part of the University of Iowa’s Summer Undergraduate Research Conference. Undergrads who were doing summer research though a UI program are required to give a poster presentation at the event, but for me it was optional, as I came in through Cornell Fellows. I knew of the event and was interested in giving a presentation. I assumed that I would be e-mailed the information when appropriate, as I was on the summer intern activities mailing list. As it turns out, there was a different e-mail list for poster information, as well as an e-mail list of summer mentoring faculty that Miles wasn’t on. By the time I figured out when the conference actually was, the registration deadline had already passed. Thankfully, the program manager was sympathetic, as others had the same issue, and although it was an extra hoop to jump through I managed to get on the schedule.

The last few weeks had been rather quiet, but week 10 made up for it. I consolidated the living FACS wells into a new plate, as described above, but before we could move on with the cell density assay, Mimi and I had to test the BCL6 gene segment qPCR primers. We used a quick extract procedure to get some NALM6 genomic DNA and check to see if we had amplification with gel electrophoresis, and we tested several other primers along with BCL6 primers. Between the two of us, we tried about 5 times with different template densities, PCR buffers, and even used qPCR to determine the optimal annealing temperatures of our primers. However, we saw either not amplification or heavy smearing/multiple bands that did not include the size of the expected target. Only later did we discover that a mistake had been made in the primer order. Although we were bound to not get any positive results, it was useful for me to get some experience with both the techniques involved and troubleshooting PCR. The timing was unfortunate, however, as I would have loved to have put successful GR deletion detection on my poster.

Unfortunately, this was the same week that the lease on my apartment was ending and I needed to figure out where I was going to live if I wanted to continue my internship past the previously scheduled end (I am, by the way), so I was somewhat distracted from lab. Furthermore there was a difference in expectations between my PI and me: Miles thought that I would generate information like I would on a paper and that he would then show me how to organizes a poster, while I thought that that I was supposed to figure out what was expected for a poster with Miles’ help before I started to produce the figures and information. Each of us was waiting for an anticipated move by the other. Between this, the apartment issue and a sudden surge in lab work, even after Miles made his expectations clearer and gave me some deadlines I managed to get behind on a project I already had short notice on. The poster was more or less constructed over the course of two days at the expense of Miles and me. This was not a pleasant experience and is a glaring blemish on my summer experience here, but it was also a learning experience about proper communication and poster construction. Also, the final product turned out surprisingly well.

The conference itself also went surprisingly smoothly. All the posters were up by 1:30 and the 145-ish presenters were split up into two groups: half presented their posters from 1:30 pm to 3 and the others from 3 till 4:30. That way, everyone got a chance to both present and look at other presentations. I was surprised by the breadth of topics covered by the research presented: they ranged from biomedical to rocket fuel to racial dynamics in early America. I feel like I presented pretty well for the most part. For the last few weeks I had been worried if my background knowledge was sufficient for the presentation, but I was able to answer every question I was asked. I also had some difficulty deciding on the timing of background information: should I try to present it all in the beginning or as it becomes pertinent to the results? I tried both and I think the all in the beginning method worked best. Aside from waiting by my poster for someone to ask a question, it was really fun. I was also set up pretty close to two other Cornellians who also didn’t get the e-mails and registered late.

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