Week 5 is no different. The gel electrophoresis machine is broken. We had to borrow equipment from the other lab. Hopefully this one is better.
We started by analyzing a gel from earlier in the week. The results were not great. The gel showed smears instead of clean, sharp bands, and the second band we were hoping to see was barely visible. Smears usually mean the DNA is messy in some way. It can happen if the digestion is incomplete, if the gel conditions are not ideal, or if the DNA itself is not in good shape. Kind of frustrating. That’s all I can say.
At that point, we started asking basic questions. Did we use enough enzyme? Was the buffer in the gel box fresh? Did the digestion have enough time? Those questions led us to rerun the digestion with changes. This time, we increased the amount of the enzyme.
One thing I learned the hard way was that the order and choice of enzymes actually matters. Some enzymes can be heat inactivated, and some cannot. Using the wrong one at the wrong step can complicate everything later. When we realized an enzyme had been used incorrectly, we had to clean up the DNA before moving forward. That is where DNA cleanup kits came in.
DNA cleanup is basically a way to purify DNA so that enzymes, salts, and leftover reaction components are removed. The goal is to end up with just the DNA in a smaller, cleaner volume. Even though you might start with something like 50 microliters, you often end up with much less at the end.
After running another gel, the results were not perfect, but they were good enough. This was an important lesson for me. In research, “good enough to move forward” is sometimes exactly that. Waiting for a perfect gel every time would mean never making progress. Once we decided the bands were usable, we moved on to gel extraction.
Gel extraction was one of the biggest new techniques I learned this week. After running the DNA on a gel, we physically cut out the bands that contained the DNA fragments we wanted. This is done carefully under UV light so you can see the bands clearly. The gel slice itself contains the DNA trapped inside it, so the goal is to pull that DNA out of the gel.
The gel extraction protocol breaks down the gel and allows the DNA to bind to a column. The gel is dissolved using a special buffer, then the mixture is passed through a column where the DNA sticks, but everything else washes away. After several wash steps, the DNA is released into a clean tube. At the end of this process, the DNA is no longer in gel form. It is back in liquid form and ready to be used for cloning.
Cutting the gel actually made me pretty nervous. If I messed up, a week’s progress would be gone. You are committing to that piece of DNA and trusting that it is what you think it is. It also takes patience, especially when you need to load and extract a lot of samples just to get enough DNA to continue.
Once the DNA was extracted and cleaned, the next big concept was ligation. Ligation is the step where two pieces of DNA are joined together. One piece is the insert, which is the DNA sequence we care about, and the other piece is the plasmid, which acts like a carrier. The ligase enzyme helps seal the DNA ends together, forming one continuous piece.
For ligation to work, the DNA pieces need to be compatible. That is why all the digestion and cleanup steps before this matter so much. If the ends are wrong, damaged, or contaminated, the ligation will not work. Ligation itself does not look dramatic when it happens. You mix the components, incubate them, and then wait. The result is invisible at this stage, which makes it feel a little nerve-wracking.
Throughout the week, there were moments of uncertainty. Samples were left incubating, and there were times when I was not even sure if everything had been placed exactly where it needed to be. That uncertainty is uncomfortable, but it is also part of working in a shared lab space. You learn to double-check, ask questions, and keep careful notes. Onto week 6 we go!
