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Week 1 - Getting Started
I am an undergraduate intern at OHSU’s Institute of Environmental Health (IEH). This summer I will be working with Hiroaki Naka in Margo Haygood’s lab. Monday morning, the first day of my internship, I met Vanessa, the internship coordinator, and the other summer interns in the lobby of the IEH. Vanessa gave us a quick rundown of the summer schedule and our responsibilities. Afterward, she gave us a tour of the OHSU campus. We got to ride the tram and see our labs, and she pointed out to us all of the good places to get food. Around eleven, we got lunch together, and then we dispersed to meet our mentors. When I got to the lab, Elizabeth (an undergraduate working in the same lab) and I got a tour of the lab. Then I met Hiroaki, my mentor, and we discussed some of the background for the research he is doing. He gave me a couple of papers to read on the bacteria I will be working with, T. turnerae, and on the methods he uses for gene mutation, including the Polymerase Chain Reaction (PCR).
Tuesday Hiroaki showed me how to perform PCR on the bacterial chromosome. This step separates the DNA strands and amplifies specified sections of the bacterial (or other) chromosome. Which section is amplified is determined by which DNA primer is added to the solution. In this case, we amplified fragments on both ends of the gene of interest. After lunch, we conducted a second PCR to connect these fragments. Hiroaki also showed me how to make an agar electrophoresis plate.
In the middle of the day, all of the CMOP interns met for a professional development seminar. Afterward, I went to the ID building and got my official OHSU badge. Now I can ride the tram whenever I want! Later in the afternoon, Hiroaki gave me a paper to read that explained the procedure, mechanism and some possible applications for the Splicing by Overlap Extension (SOEing) PCR method which we had used earlier.
On Wednesday we set up an agarose gel electrophoresis with the upstream, downstream and combined DNA fragments. We also included a well with DNA molecular weight marker solution. The electrophoresis process took about forty minutes to run, so while we were waiting I read a paper that Hiroaki gave me which covered the protocol for DNA extraction from agarose gel.
Once the stain was about three quarters of the way down the gel block, we turned off the machine creating the voltage difference and took the gel block out of the buffer solution. Hiroaki showed me how to use the gel viewing machine which sends UV rays through the gel to highlight the bands of DNA. First, we used the one in the lab which sends a photo of the gel and bands to a computer for viewing, analysis and storage. Then we took the gel block upstairs to an open machine which is used when cutting the agar. Hiroaki showed me how to cut out the appropriate bands so we could extract the recombinant DNA.
Back in the lab, we used the centrifuge and several solutions to extract the DNA from the gel and isolate it. Then we started the ligation process to combine the PCR product with a T-vector plasmid. This reaction takes quite a long time so we put it in the cooler until tomorrow.
After lunch, we mixed a broth to make LB agar plates which we will use tomorrow. The broth has to be sterilized in the autoclave before it is made into plates, so I got to learn how to use the autoclave machine.
Thursday morning, I had time to work on my own and read some of the papers Hiroaki gave me yesterday describing the protocol for several of the upcoming steps. Around noon all of the CMOP interns met for another brown-bag seminar, this time covering research ethics.
In the afternoon, Hiroaki showed me how to transform the E. coli with the plasmid we ligated yesterday. The process took a few hours because there are several steps which involve incubation time. Once this was done, we plated the transformed E. coli onto the LB agar plates that we made yesterday.
Friday morning, I got to the lab a little earlier than usual. I worked at my desk for a little while until Hiroaki arrived. Once he got there, we went to the 4oC room and got out one of the agar plates that we had prepared on Wednesday. While the plate was warming to room temperature, we prepared a beaker of sterile water and put it in the autoclave machine.
When we got back from the autoclave room, the plate was ready, and Hiroaki showed me how to transfer individual colonies from the agar plates we prepared yesterday to the new plate. The two plates we prepared yesterday now contained both white and blue E. coli colonies. The white colonies contained a plasmid which included both the T-vector and our PCR product fragment. The blue colonies contained only the T-vector plasmid. We selected around twenty five isolated white colonies from the two plates combined and transferred them to the new plate using toothpicks. Hiroaki explained that such a large number of colonies is needed for extraction because gene sequencing can sometimes require quite a few samples to get accurate results. In addition, some of the plasmids may mutate in the E. coli and will no longer be useable in the experiment.
