Week 8 felt like the home stretch. With school starting again next week and the Moving Targets Symposium set for 8/21/2025, the pressure was definitely there.
The first task of the week was sequencing. The miniprep results from last week came back, and unfortunately, most of them weren’t right. On Friday (8/1), Teddy ran my sixteen colony PCR samples on a gel to check which ones looked the strongest. Looking at the gel, we picked two colonies that had the clearest, strongest bands: Colony 3 from the new 5 µl ligation and Colony 3 from the old 5 µl ligation. These were the best candidates to send off for sequencing.
When the sequencing results came back, there was a moment of relief: Colony 3 of the old 5 µl ligation sequence was correct. That single successful colony meant everything else could finally move forward. From here, all work focused on expanding this colony and preparing enough plasmid for experiments. Another round of bacterial transformation was done using just this verified sequence. The bacteria went into the shaker to recover, then were plated and left overnight in the incubator.
The next day, colonies had grown, and five were picked to start primary cultures. These were placed into the shaker for about sixteen hours, giving the bacteria time to grow and multiply. After the primary cultures, the next step was to prepare for what’s often called a “secondary culture,” which in this case was culturing bacteria for a midiprep to extract larger amounts of plasmid DNA. You’ll notice we often do the same thing over and over again. Research is pretty repetitive but every experiment yields different results, so it is still quite interesting.
At the same time, we needed to make sure the bacteria we had worked so hard to grow were stored safely so we could use them later without having to start all over again. To do this, we made a special freezing mixture using 50% glycerol and 50% ultra-pure water. Glycerol is like a protective liquid that keeps the bacteria from getting damaged by ice crystals when they are frozen. For each colony, we took 500 microliters of the bacterial culture and mixed it with 500 microliters of this glycerol solution. Then, we carefully put these mixtures into tiny tubes and froze them in the -80°C freezer. This is super cold and it basically puts the bacteria in “pause mode,” so they can stay alive for months or even years without growing. We made two separate tubes for each colony as a backup, just in case something went wrong with one of them. That way, we always have a safe copy of the verified colony that we can use in the future, so all the work we did growing, checking, and sequencing the bacteria isn’t lost. You can come back later and pick up exactly where you left off.
With the sequencing confirmed, all that hard work finally paid off. This correct sequence would be the one used in experiments moving forward, and it would also be the image featured on our poster for the symposium.
Even though the last week was still busy, there was a sense of satisfaction in seeing things finally come together. It wasn’t just about finishing experiments; it was about understanding the process, learning from all the mistakes and retries, and having a sequence we could trust. Now, all I have to do is create the poster and prepare to present. I’ll make another post regarding the symposium soon!
