Student Research at Duke

I’ve never really thought about any other career choices other than in medicine, but after the panel talk last week, I think my options have broadened up a bit. A joint MD-PhD sounds pretty interesting to me and I’ve thought about the prospects of doing research in medicine. Likewise, I think going into the private sector to conduct some research before going on to become a doctor is pretty practical as well. Not only do you get some experience outside of residency and fellowship, but the research techniques and experiences last forever. As for a career in the pharmaceutical industry or elsewhere, I don’t think that would be my cup of tea. I’m more interested in the daily face-to-face interactions of medicine rather than the behind-the-scenes work of large industry.

But maybe I’ll give it a try. You never know.

As this week comes to an end, several things have changed…for better or for worse.

The good news: My PCR modification succeeded in that the primers didn’t degrade before the transformations. I suspect that during the first few weeks, the annealing temperatures were too high, and since our primers had relatively low melting temps, they “died.” Also, the transformation into BL21 cells for protein overexpression worked as well, as I got confirmation from my protein gels. Now, those samples are purifying in the FPLC. Very Nice. So now, four out of the eight point-mutagenesis trials are working.

The other four…have problems. One mini-prepped DNA sample came back negative for point-mutagenesis, so I’m going to redo that sample. Another three plates didn’t have colonies, so I might redo the PCR for those primer samples and restart the whole procedure. If that doesn’t work, I’ll probably lower the PCR temps again. We’ll see.

On a side note, I’m really excited for the last few weeks of my fellowship, as it’ll be time to analyze all the data that I’ve gotten so far. And just today, I’ve received those DNA sequences for the mini-preps. Next week will be killer, as I’ll have to start on an abstract and poster design for the culmination of this program.

The week of Fourth of July is almost over, and luckily, the final preparations for one of my samples is almost done. I’ve been finishing up on a PCR modification (since our annealing temperatures were initially too high) and by next week we’ll hopefully get some results. As for the sample that did work, it’s been sitting in the incubator/shaker.

By next week, I’ll be able to retrieve the cells that overexpressed our modified ubiquitin. After that comes protein purification and SDS-PAGE gels.

Next week’s going to be busy.

This post starts off the second month of research under the HHMI program here at Duke University. In the previous month, I got to know my PI, my mentor, and the lab group, as well as various lab techniques. Cell cultures were started and we ordered primers for point-mutagenesis of ubiquitin. Now, in the second month of research, I’ll hopefully be transforming competent cells and overexpressing our modified ubiquitin to determine specifics about its structure and modes of function. But, as of now, certain PCRs don’t work and our sequencing data came back negative.

“Negative, Mr. Lian.”

But, Fourth of July week is upon us and the lab has quieted down somewhat, but for me, there is still work to be done.