Day three was full of gels, gels, and more gels. I felt much more comfortable today due to my acquired knowledge about gels from biotech class. Even though I was familiar with the process for running gels, there were still some differences between our Biotech protocol and Dr. Bermek’s protocol which allowed for so much learning. Preparing the gels one on one also helped me learn and understand so many new things about them because I didn’t have any lab partners to rely on. I started the day similar to yesterday with a run down about the results from the experiments that were pending. Once I heard the results, we made a plan for today. The first thing we did was run a protein gel. I was vaguely familiar with this type of gel because of our recent fish project in biotech. I really enjoy Dr. Bermek’s warm and comforting personality. When I make a mistake she’s really nice about it. I really appreciate how she lets me feel like I’m involved with each experiment while giving me just the right amount of help I need.
First, I added 4 uL of 6x dye to tubes labeled 1-9. Then, I added 14 uL of each sample to the various tubes. Once the samples were ready to be loaded, she walked me through the process of setting up the protein gel. Unlike an agarose gel, the gel we used for protein electrophoresis was already prepared, so we didn’t have to pour it. We retrieved the ready-made gel from the refrigerator. We put on gloves for this specific gel because it needed to be handled with care. We took it out of its package and removed the sticky strip of plastic on the bottom or else the electric currents would not run through it. We also removed the comb from the top, so we could load the samples. Then, we placed the gel on one side of the tetra gel box. We made sure it was firmly placed into the gasket so there were no leaks. Parallel to the gel, we put a sheet of plastic so the liquid TBE buffer would be contained. When placing the piece of plastic I learned it was important to have the words “buffer dam” facing me. Dr. Bermek showed me some tricks when setting up the gel like putting it on the side closest to the edge of the gel box so the samples would be easier to load. Once we had the gel rig all set up, we placed it in the tetra gel box with the red cathode matching the red sticker on the box and the black anode matching the black sticker on the box. Once the whole box contraption was set up, it was time for loading the samples. I was extremely nervous because, for me, this was the hardest gel to load. The wells were so small and samples could easier drift into another well causing unwanted contamination. With Dr. Bermek’s wonderful teaching approach she showed me how to load the first three wells and then gave me a chance to do the last 7. I was doing okay on my first two, but when I got to the third one I didn’t place my pipette in at the right angle and some of my sample got into the next well. I informed Dr. Bermek of my mistake and she was really understanding a offered an easy fix. She pipetted out the contaminated well and finished loading the rest of the samples. Once they were all loaded, she turned on the electric currents. Unfortunately, 30 minutes later we saw the samples swimming in the buffer. We made a common mistake when turning on the electric currents because the black and red wires got mixed up. Since we realized 30 minutes into the gel being run, we couldn’t salvage the samples.
We repeated the experiment again, but this time, I did it all almost entirely by myself. I setup the gel rig and box under Dr. Bermek’s eye but I physically did everything. I then, repeated mixing 9 more samples, so they would be ready to load. I incubated them and spun them all by myself. Then while Dr. Bermek was checking on another gel. I loaded all 10 wells by independently with only a few small mistakes. I was very proud of myself. I won’t know until tomorrow how the gel turned out because it needed to be stained for a long time, but I’m hoping everything worked out well. Once we had finished repeating the protein gel, Dr. Bermek’s agarose gel she had done the night before had finished staining, and she showed me the results. The reason she did this gel was to compare the work I did with the electron microscope yesterday. She showed me the gel image and began explaining and annotating it. The first lane there was just plain DNA which was the control. It had a lower molecular weight, therefore it was super coiled. This gel confirmed our results we obtained from the electron microscope yesterday. The second lane contained the UL8 protein which relaxed the DNA therefore the band was at a higher molecular weight. The third band had UL8 and CIP. UL8 relaxes the DNA but CIP supercoils it, so the band had a lower molecular weight meaning the DNA was supercoiled showing us the CIP was working. The fourth lane had topoisomerase which again relaxes the DNA therefore the band was at a higher molecular weight; however, lane 5 did not produce results we wanted. Lane 5 had topoisomerase and CIP, but the band had a higher molecular weight meaning it was relaxed and not supercoiled even though CIP is supposed to super coil the DNA. We suspected that since the topoisomerase she used was a different brand than normal it might require more CIP than the UL8 needed. The next 6 lanes were treated with different amounts of UL8 which had all the bands at a lower and higher molecular weight meaning they were intermediate DNA (some parts supercoiled and some parts relaxed). It was cool to see what intermediate DNA looked like through the EM and on a gel. The last 5 lanes had topoisomerase and Ethidium Bromide. The topoisomerase relaxes the DNA whereas the Ethidium Bromide is supposed to super coil it, but again, our results didn’t quite match what we were expecting. I learned so much about the function different proteins have on DNA. It was cool to compare and contrast my findings on the EM and on Dr. Bermek’s gel. I like how in science you can do so many different tests and learn about the functions of things. After our day full of various gels, we topped it off setting up one more agarose gel to use for tomorrow. I really enjoyed today. I got to work with techniques I was familiar with, but still learn countless new things about gels. I also learned so much, just from, hearing about and annotating the gel image from yesterday. I don’t know what’s in store for tomorrow, but I can’t wait to get some of our results and continue preforming experiments.
Informative annotated Agarose gel.
Tetra gel box setup and ready to be run.
My beautifully loaded wells!
Our sad samples swimming in the buffer after our mistake.
Second times the charm! The correct box with the samples down at the bottom of the gel instead of floating in the buffer.