October 29, 2021
Marley Fuhrman
Last semester my lab partner Abby Mueller and I successfully created a plasmid with the downstream insert of our SprB gene and named it pFM01. This semester I’m continuing research by inserting the upstream gene insert into the pFM01 plasmid to make what I’ll call pFM02, which is the final plasmid that I am working towards. This semester is quite a bit different than last because my lab partner transferred schools and I am continuing my research without her. This created new challenges for me this semester by forcing me to learn to work on my own (for the most part) and to not rely on a lab partner to help with projects or even normal daily lab tasks. This humbled me a bit and made research a little more difficult this semester.
Specific Content:
To start this semester, I needed to do a Phusion PCR with the upstream DNA of my gene that showed a band, which took the longest because I kept running into issues with my gels. The band wouldn’t show up, or something with the gel would go wrong. To try to get an expected PCR product, I tried different annealing temperatures in the thermocycler when making the Phusion PCR, and I tried doing a gel extraction to get non-specific bands to disappear, but neither worked well.
Figure 1. Marley waiting for one of the many gels she made to finish running.
A few weeks ago, I ended up trying to make my Phusion PCR with a new primer, and it worked! The new primer made the Upstream DNA show a band in the PCR which allowed me to continue working on my research. Another hiccup in my research was that the concentration of my pFM01 plasmid wasn’t good. To solve this issue my lab TA and I prepared more plasmids from the E. coli obtained last semester, digested and purified them, then ran a sample in a gel to see if a band would show, and it did.
Plans and Conclusions:
Next, I will be doing ligation of my upstream insert with the pFM01 plasmid and transformation. Then to see if it worked, I would grow E. coli samples and test the plasmids in the grown colonies to see if both the upstream and downstream inserts are fused in the plasmids. My research this semester may not be going exactly how I planned, but I’m finally getting the results I’ve been working towards for over a month and now I can continue trying to create pFM02, so things are looking up.
