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Week 3 - It All Starts to Make Sense
This week was really exciting for me because a lot of the work that we had been doing up until this point began to make sense. On Tuesday, Hiroaki sent out the first gene fragment for sequencing and on Wednesday I got to start my first solo reaction process.
Monday morning Hiroaki gave me a couple of journal articles to read which discussed the function of a biosynthetic gene cluster in Teredinibacter turnerae (the bacteria I am working on) that codes for molecules which aid in the functioning of siderophores (structures which help with iron binding). Part of my project will involve trying to confirm the proposed function of a section of this gene cluster, so these papers gave me some background understanding for the work I will be doing. In the afternoon we transformed E. coli with the two gene fragments we had been working with last week.
Tuesday was a field trip! Today we visited the Bonneville Dam. On the way we stopped at the Vista House and the Bonneville Fish Hatchery.
When I first got to the lab on Wednesday, I read a two journal articles that discussed the protocols for several of the methods we have been using so far. Afterward, I started PCR for the third gene region that we are investigating. Hiroaki already had primer solution mixed for this fragment, so I just had to make a diluted walking solution. Then I prepared the upstream and downstream solutions and started the first PCR. Once that was started, Hiroaki showed me how to pick colonies from the transformed E. coli started on Monday. I picked twenty white colonies and streaked them individually onto a new LB/Ampicillin agar plate. Then I put the plate in the incubator to grow overnight. After that, I had a little bit of down time until lunch because the PCR reaction was still going. After lunch I started the second PCR reaction and then read more journal articles while the reaction was running.
Thursday morning I made an agarose gel block when I first got to the lab and then I started an agarose gel electrophoresis with the PCR products from yesterday. Once that was running, I took the E. coli plate that we made yesterday out of the incubator, wrapped it in parafilm and put it in the 4oC cooler. After that was done, I still had some time until the electrophoresis was finished so I did some reading and work on my computer. Once the electrophoresis finished, I took the gel out and imaged it on the lab gel camera. Then I took the gel upstairs and placed it on the UV light to cut out the gel strip containing the second PCR fragment. Afterwards it was time for lunch. In the afternoon, I extracted the DNA fragment from the agar gel strip and then started a ligation reaction. This took a few hours, so when I was done I had time to read journal articles before I left for the day.
Friday was a holiday (the fourth of July!) so there was no work.
