This was our first week in the wet lab, and the team was excited to begin working! Though we were still waiting for the plasmids to arrive, we made the most of our time by diving into lab orientation. We explored the lab, learning where everything was—from waste bins and PCR tubes to the water bath, pipettes, and biosafety cabinets. Alongside this, the team practiced sterile techniques like inoculation and aseptic handling of both liquid cultures (LB broth) and solid agar plates. We also got hands-on experience working in the biosafety cabinets, where we’ll be handling bacterial cultures moving forward. It was a great chance for everyone to get comfortable in the lab, ensuring that when the plasmids arrived, we would be ready to hit the ground running!
The following Plasmids from AddGene arrived: PMevT, PJBEI-6409, and PMBI.
Today, we focused on creating cryostocks of bacteria—essential backups in case of contamination. Simply freezing the bacteria is not a viable option, so we added 40% glycerol to cryopreserve the cells. Using a 1:1 ratio of bacteria to glycerol, we made 1ml aliquots and stored them in the –80°C freezer. While this occurred, we performed plasmid isolation using the NEB Monarch Plasmid Mini Kit Prep. Since we no longer intended to propagate the bacteria, we were able to conduct the isolation in a non-sterile environment. The process began with centrifuging vials containing our bacteria and media at 3200 RCF for 3 minutes, yielding E. coli pellets. After discarding the supernatant, which consisted of excess media, we focused on the pellets! Our results from plasmid isolation were promising: PmevT: 23.5 ng/μL PJBEI-6409: 14.6 ng/μL PMBI: 9.4 ng/μL PMBI (repeat): 22.5 ng/μL With these concentrations, we had enough isolated plasmid to conduct all of our future experiments! The samples were frozen and ready for use next week.
For our first day in the lab this week, we focused on making agar plates to conduct transformation. We transformed E. coli with one of our plasmids and plated the culture, which is essential for our experiments. Additionally, the team prepared a menthol serial dilution. We reconstituted all the primers we received last week to a concentration of 100 µM and then diluted the forward and reverse primers to 10 µM to use in our PCR experiments. The excitement was palpable as the team prepared for PCR, carefully ensuring all necessary components were pipetted into the PCR tubes. After initiating the PCR process, our next steps were to proceed with gel electrophoresis.
On the second day of lab, everyone learned how to make agarose gels! However, a slight mistake happened- instead of TAE buffer, we added distilled water, and the gel electrophoresis did not run properly. It was a great learning experience, and we will not make that mistake again! Later, a second gel electrophoresis was run with the correct reagents, and our results were promising.
Since today was our first and only day in the lab this week, the team focused on setting up DNA gels for the electrophoresis and making agar plates. To make the DNA Gel, 1% TAE Agar Gel was produced by adding 5g of agarose powder into 500 ml of TAE buffer and then heating this mixture until it boiled. Once done, 5 uL of Cybersafe was added to 50 ml of gel solution. This solution was then poured into the gel casting tray with a comb added subsequently. The gel was then left to set for 20 minutes. While the gel set, 18 agarose gel plates were made: 6 with Tetracycline, 6 with chloramphenicol, and 6 with no antibiotic as a control. This was done by measuring out 150 ml of DI water, which was poured into an Erlenmeyer flask. 6g of LB Agar Miller was then added to the water and subsequently microwaved in 30-second intervals until the powder fully dissolved and bubbles formed. While waiting for the solution to cool, the plates were labeled using the following template: LB/antibiotic_ initials_ date and then 150uL were added to each labeled plate. Although these plates were not used today, they were prepared for future experiments to validate successful transformation of linalool and menthol plasmids.