Week 3: Molecular Work

In my third week, I continued developing my proficiency in the lab. This week I focused less on the sediment coring and more on molecular techniques that will allow me to quantitatively analyze our sediment samples. I got very comfortable with extracting DNA from sediment samples, as well as analyzing its concentration using the NanoDrop, making series dilutions and updating the Simon lab Excel sample sheet we use to keep everyone up to date on our sample processing. Furthermore, I attempted my own qPCR-which stands for quantitative polymerase chain reaction- plate after watching Kiley do a few of her own. We used specific primers to search the sediment samples for a significant presence of the AmoA gene, which is the gene that ammonia-oxidizing archaea (AOA) use to digest ammonia. The presence of high amounts of the AmoA gene in sediment fractions where snails were found could indicate that the native archaea are responding to the increased concentration of ammonia (due to the snails) by upregulating AmoA transcription and therefore their ability to uptake it. I found doing my own qPCR a bit overwhelming at first. It requires precise pipetting, and it can be difficult trying to keep track of several samples at once. However, my results and PCR efficiency looked decent, so I am looking forward to continue building my skill set. I learned not only how to make the physical PCR plate, but also how to set up the computer to run the PCR program itself, as well as how to export and analyze the data on Excel on my computer to get a better idea of the results. Some of our initial runs looked a bit odd, with peaks in fractions without snails, so I also did a DNA purification protocol on all of the samples. The results even after clean-up were a bit confusing, so we are going to continue testing samples next week.