Week 3: qPCR

After recieving the sequence information of my two plasmids, I found both to match the database sequence I was attempting to amplify. Knowing the number of basepairs in the sequence then allows for the calculation of the number of copies of the sequence in a known volume of solution. This is necessary information to run qPCR on a sequence as standards are required to create a concentration curve for which unknown samples can be compared to in order to quantify the presence of a gene. Serial dilutions of the plasmid containing solution were then prepared as standards and initial qPCR experiments were underway. I soon learned that qPCR is a very sensitive and tricky technique to perform. My first few attempts resulted in unusable standards that did not form the linear trendline that is expected. This was probably due to incomplete mixing of solutions before filling wells. So over the course of the week I worked on improving my ability to set up a qPCR well plate with Ian's help in order to get interpretable results. The initial data from these experiments indicates that the Gyromitus USE sequence is present at higher frequencies in the freshwater of the Columbia River as opposed to the marine environment.

    In addition to my attempts at qPCR, I worked to find a general primer that could be used on other Cercozoa species for which I had primer sets that were not suitable for qPCR. The main problem was that these primers resulted in amplicons that were below the recommended 100 bp. Attempts to design specific primers were difficult due to the small amount of sequence information avaible. So I will need to work on generating an expanded USE sequence for many of the CRE Cercozoa in order to design specific primers. Furthermore, I also designed some specific primers for the USE of a variety of Cercozoa species identified in the Vancouver, BC area. These primers will be used to determine if those same species are present around the CRE as well as Vancouver in order to demonstrate the wide distribution of these organisms.