The three colonies from Week 1 had grown overnight, so Week 2 started with spinning down those bacterial cultures and extracting the plasmid DNA. I used minipreps for that step. I had done minipreps last summer, so the procedure itself was familiar. What mattered this time was what each tube represented: one candidate version of the FLT3-TKD D835Y plasmid that might or might not contain the mutation we were trying to make.
A miniprep separates the plasmid DNA from the bacteria that copied it. We needed that purified DNA before we could measure it or send it for sequencing. After finishing the extractions, I used a NanoDrop to measure the DNA concentration in each sample. The three colonies did not give us equal amounts. One candidate was too low, while the stronger samples had enough DNA to move forward.
That difference mattered because a sequencing sample needs a usable amount of clean DNA. Too little DNA can produce a weak read, which would make it harder to tell whether the D835Y mutation was actually present. The NanoDrop number did not tell us whether a colony was correct. It only helped us decide which candidates were worth sending.
The newest part for me was designing the primers for Sanger sequencing. Last year, I usually worked with primers that had already been chosen. This time, I had to think about where the sequencing read needed to begin. A Sanger primer is a short DNA sequence that gives the sequencing reaction a starting point. From there, the machine reads the bases that follow.
The goal was not just to get any stretch of sequence back. We needed a clear read across the part of FLT3 containing the engineered D835Y site. That meant choosing a primer that matched the plasmid in the right location and pointed toward the region we cared about. If the primer started too far away, the quality could get worse before the read reached the mutation. If it started in the wrong place or direction, the result might not answer our question at all. Designing it made me understand that sequencing depends on how you ask the question, not only on the machine that returns the data.
Once the primer was selected, I calculated the plasmid and primer amounts for the sequencing tubes. We chose the stronger candidate samples based on their concentrations and prepared each one with the corresponding primer. The calculations were less interesting than the primer design, but they still determined whether each tube contained enough DNA and primer for the sequencing service to return a readable result.
Then the samples were sent out for sequencing. After two weeks of PCR, bacteria, overnight cultures, and purification, the next answer was out of my hands. There was no extra step I could do to make the mutation appear. Either one of the colonies contained the correct D835Y sequence or it didn’t.
So Week 2 ended without a final result. We had narrowed the candidates, designed the primers, and prepared the samples carefully enough to get a useful answer. Now we had to wait for the sequence. Week 3 would start by finding out whether any of that work had produced the plasmid we needed.
