Part 2. I want to try to explain the first two experiments I ran without randomly throwing scientific jargon. It was finally time to learn by doing.
The first experiment I did was PCR plasmid confirmation. If that sounds like a mouthful, here’s the simple version: PCR (Polymerase Chain Reaction) is basically a way to make a ton of copies of a specific DNA piece, in this case, the FLT3 mutation that’s important in AML. Think of it like photocopying one tiny piece of DNA millions of times so we can actually see it and work with it later. Without PCR, it’s like trying to read a book that has only one letter written on a huge page… guess what? You’d never see it.
Before we started, we talked about controls. Controls are like safety checks to make sure the experiment is working the way it’s supposed to. We had two:
A positive control, this is a DNA sample we already know has the FLT3 mutation. If this works, it tells us the PCR reaction itself is functioning correctly.
A negative control, water with no DNA. If this shows up positive, it means something is contaminated or went wrong, because water should have nothing in it.
Setting up the PCR mix felt a bit like cooking a complicated recipe. You need to calculate how much of water, buffer, and enzymes you need. Everything goes into a big eppendorf (big tubes) called a mastermix before splitting evenly into a predetermined amount of PCR tubes (small tubes). Once the PCR was set up, it was just a matter of putting it in the thermocycler, which is the machine that heats and cools the DNA to make copies.
The next day we did gel electrophoresis. I had my welcome to research moment. My first try was a total disaster. I loaded the samples into the gel, turned on the voltage, and when I came back all the line had disappeared. There was not even a faintest line at the bottom (which is what would normally happen if you ran the gel for too long).
Gel electrophoresis is a way to see DNA by separating it based on size and making it glow. In the lab, we started by mixing the gel powder with TAE buffer and microwaving it until it became a liquid. We swirled it to make sure everything was fully dissolved, then let it cool just a little so it wouldn’t melt the ethidium bromide. Ethidium bromide is a chemical that binds to DNA and allows it to glow under the iBright machine later on. Once the gel was cool enough, we added the ethidium bromide and poured the mixture into a mold with little wells at one end. We let it sit until it hardened completely. After that, we carefully loaded our DNA samples into the wells, along with a DNA ladder, which acts like a ruler to show the sizes of DNA fragments. When the electricity was turned on, the negatively charged DNA was pulled toward the positive end of the gel. Smaller DNA fragments could move through the holes in the gel faster, while larger fragments moved more slowly. After the run was finished, we put the gel under the iBright machine and saw glowing bands. Each band represented a group of DNA fragments of the same size. By comparing the bands to the DNA ladder, we could tell if our DNA was the right size and if the experiment worked. It is a careful process that requires patience, but seeing the glowing bands appear is really satisfying because it shows the results of all the work that went into preparing the samples and the gel.
Another thing I learned was just how long each experiment takes. An experiment usually takes 3-4 hours. A lot of that time is waiting though. For example, the thermocycler usually takes about two and a half hours to finish a run. Throughout these two days, I realized how much research is a mix of patience, precision, and problem-solving. On paper, it sounds straightforward: amplify DNA, run it on a gel, confirm your results. But in reality, every tiny factor affects whether the experiment works. And if something goes wrong, you have to think critically about what caused the issue, troubleshoot, and try again.
My brain feels fried. In a good way.
