Blogs

This week with Tuesday being a day off from work to tour the Bonneville Dam and Friday being the 4th of July only consisted of 3 days in the lab. I recieved a new batch of primers on Monday that were designed to amplify USE from Cercozoa isolated in Vancouver. So Wednesday and Thursday I ran a few PCR reactions on samples from the CRE as well as La Push as a potential positive control. I was hoping La Push, the closest total extract I have to Vancouver Island, would contain some of the same Cercozoa but unfortunately the results of PCR indicate that it does not.

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. 

This week was a short week due to Independence Day and a field trip, and the week was mostly spent trouble-shooting. On Monday, we tried something new to be able to read the 4-chloroaniline concentration on the HPLC. Our conclusion as to why we were seeing no 4-chloroaniline in our HPLC output was that the aniline was adsorbing to MnO₂, so when we filtered out the MnO₂, we also effectively filtered out the 4-chloroaniline.

I continued to study the manganese ligand solutions this week. We are waiting for one last chemical to come in before we can start measuring superoxide, but we are hoping to be able to begin that next week. I learned more procedures for making manganese solutions, including manganese pyrophosphate, which is a pink solution, and manganese desferoxamine, which is dark green.

When I analyzed last week's qPCR data, I found that the efficiency was only 85%, which was below the value we were hoping for. So this week I got to practice some more qPCR, and hopefully when I analyze the most recent data, my efficiency values fall in a more acceptable range.  We also started planning for RNA extractions next week, which involve a lot of solutions that need to be prepared, so I took the time to plan out all of the solutions and calculate all of the necessary volumes and such.

One of the highlights of the week was receiving this email from my PI:         

Though this was a short week due to the 4th of July, it was a very busy and really fun week. On Monday, Claudia returned from her week off, and so I brought her up to speed on my project's progress. I then helped her prepare for our sampling trip to Camas to collect water and nutrient samples from the Columbia. I am definitely getting comfortable with the field work we are doing and really enjoy our time on site. After we got back from sampling, I helped to process the samples and analyze the nutrients.

I entered my second week raring to go, ready to work as hard and as fast as I could. I wanted results, and I wanted them NOW. After three intense days, I end this week feeling very different. I care more about doing my work carefully and correctly, even if it means spreading my work over more days. I also care more about taking care of myself, since this neglect made me susceptible to making mistakes.

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.

After recieving an order of primers I am now able to work on amplifying the USE of some of the Cercozoa organisms I am studying. The initial PCR experiments I ran were attempts to amplify DNA from three 28S unique sequence elements that closely matched species in the Bodomorpha and Gyromitus genera as well as an unclassified Cercozoa species. The Bodomorpha primer appeared to amplify LSU sequences with little specificity as evidenced by a smear of DNA visible after running a gel of products.