Social
This week social wise started off with social hour with a large group of interns. Social Hour consists of one of the interns sharing a presentation about their home country. This month it was Pakistan. I was really intrigued by the vast sites filled with beautiful views. We also were able to try traditional Pakistani foods which were excellent. Later in the week, the IRRI interns played volleyball with other interns which was a blast. During the weekend we hiked Mount Makiling which was definitely an experience. The mountain was full of leeches which were unpleasant, to say the least. By the end of the hike, I made the decision that just not looking till the end was better and pretending they weren’t there. The view was incredible though and the flora alone was amazing. Toward the top, it was a lot of climbing but everyone made it through okay. After the hike we went back, I took a very long shower and took a much-needed power nap. We went out with Hunter and Sam later that night and showed them some of our favorite spots in the town.
Research
Well, it happened. The dreaded moment all my Professors have warned me about. The point where your experiment doesn’t work and you have no idea why and just have to keep trying. The whole week I got no amplification of my markers. I was having trouble the previous week so going into this week I threw everything I could think of (shout out to Ate Pat, Ate Grace, Ate Lots, and all the others for all the help) to fix it. First, I used the Nanodrop to check the DNA concentration which came back normal. Our first test yielded very confusing results or lack thereof. I tried to isolate every variable in some way. I tried to separate DNA and different markers that were used in a previous experiment and “knew” worked. I isolated each possible PCR mix variable and tested the different PCR components I used. I then used different DNA samples that were used for my markers and tested each of those with previous markers that worked and the markers that haven’t been working. I also had another experimental group within this level which also tested PCR products. Just to be sure there were no errors in protocols or how I carried out the procedure we reviewed those as well. No errors were discovered in the procedure. I ran the PCR and then the gel. The results…. no amplification. One of the others in the lab said the same thing happened to them despite the Nanodrop reading adequate concentrations and extracting new DNA resolved the issue. This would not explain the other DNA not amplifying, though. So, the following day I went to the glass house and retrieved new samples. This consists of finding the second youngest leaves on the plants of the 16 species I am comparing. It also involves sweating through the clothes you have on. I brought the samples back to the lab and then returned to my dorm to shower and change. After lunch, I extracted the DNA and ran the PCR/gel which, once again, had no amplification. This test was with all markers that I knew worked but were markers within my experiment. The next day I came into lab and ran the experimental setup I tried on the first day with the new DNA. I also used a different PCR machine. Once again, no amplification. In the afternoon, I ran it once again but this time I used different loading dye because at this point, might as well try it because nothing else worked. In this round, I set up the plate to differentiate between 3 DNA sets, and 3 PCR components, one of which was premixed by another lab member and they used that day which worked. The results. Everything amplified! I was very relieved while also a bit annoyed that the issue was the easiest thing to fix and the one variable we didn’t consider. This was what I thought to be a big lesson for me to read and record carefully because I missed the fact that I used a new loading dye the first day the issue started happening. Yet, I went into lab the next day, and… no amplification. I ran a couple of PCRs and got some amplification but not to the extent where the results could be scored. We will see what we can do next week and hopefully resolve the issue. I will say, I have learned a huge amount this week about the experimental process and understanding how to isolate variables which as grueling as the week was, I am happy about.
Pictures
