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Week 2 - Getting into the Flow
This Monday we started two more PCR reactions. Hiroaki did one of them to remind me of the steps, and I did the other. The new primer that we needed came in over the weekend, so the first step was to prepare the primer with autoclaved water. Then we diluted a portion of the primer to a walking solution and used that to prepare the PCR solution. PCR takes around two and a half hours, so after we started the reaction we took a lunch break. In the afternoon, we started the second PCR reaction which will run overnight. Then Hiroaki showed me how to inoculate tubes of LB broth with the bacteria from last week.
On Thursday morning I prepared an agarose gel solution and poured it into the electrophoresis mold. Then Hiroaki showed me how to extract the plasmid from the E. coli that we put in the broth on Monday. While I was gone, Hiroaki had isolated 3 ml of solution from each of the six tubes and centrifuged it to form a pellet of E. coli cells. We used a kit from QIAGEN to re-suspend the cells, lyse them, and then extract the plasmid using the centrifuge, a DNA filter and several specialized solutions. A final buffer solution was used to remove the plasmid DNA from the filter, and then the solution was stored in the 20oC freezer until Monday when we will send a sample out to be sequenced. At noon, I went to the all OHSU internship party with the other interns from my lab.
In the afternoon, we started the agarose gel electrophoresis with both sets of PCR products from Monday. While we waited for the electrophoresis to finish, I looked at an article that Hiroaki recommended that explained a new method of genome sequencing which Margo and her colleagues used to sequence the T. turnerae genome.
Once the electrophoresis finished, we used the gel imaging machine to look at it and then stored the picture on the lab computer. Like last time, we then took the gel up to the open machine to cut out the second PCR product. Then we used one of the QIAGEN kits to extract the DNA from the gel.
Friday morning I got in a little early and read a couple of articles Hiroaki suggested yesterday that discussed the new generation genome sequencing methods. When Hiroaki got in, we started the ligation reaction for the two chromosome regions that we started working with this week. The tubes containing the reaction went in the 4oC cooler to react over the weekend. Our schedule got a little off for this week because I was gone Tuesday and Wednesday, and as a result, there was not a lot that needed to be done today.
After we finished, I went over to the Hatfield Center to talk to Vanessa about information that was covered in meetings on Tuesday. When I got back, I had a little bit of extra time before the lab meeting, so I did some research on my own about the DNA extraction process we have been using. At one, the whole lab gathered for a lab meeting and pizza! Also at the lab meeting, we got to listen to a speech in the works by one of the members of the lab who is preparing for her defense in a few months.
