Today was full of learning new techniques and exciting experiments. Dr. Bermek and I spent the first half of the morning discussing the experiments she still had to complete before her paper about the Herpes Simplex virus could be written. We decided to perform an experiment that could potentially lead to never seen before results. There is a shape of DNA that people have not been able to get, but with the protein UL8 we have been working with, Dr. Bermek wondered if this protein could help make the DNA shape because UL8 relaxes DNA and creates a nicked structure. Dr. Bermek wasn’t super optimistic we would get results but she wanted to try anyway. A large experiment like the one we were about to do calls for a lot of controls because, if it works, we want to make sure we know exactly what helped create the desired DNA shape. We set up 7 reactions that each included or didn’t include a specific ingredient. For example, one sample didn’t have UL8 but had UL30. The reason is because there has been speculation that UL30 might cause DNA to become nicked, so we wanted to have it as an option, but if it worked, we wanted to make sure that it was the UL30 protein that caused the DNA shape not the UL8.
Dr. Bermek had me make the master mix which is basically a large volume of ingredients that needed to be added to each reaction. It is used to save time and eliminate unwanted errors. I was happy she trusted me enough to make it. The timing of the preparation of the samples was overwhelming and difficult. We had four different timers going all for different things. We had to add specific ingredients to some of the seven samples but not others in addition to making sure the timing was perfect. Some samples had to be heated for five minutes others had to be heated for 30, and there were other parts of the experiment that required keeping a close eye on the time. Dr. Bermek and I had a good system going to help everything run smoothly. She would take the samples out the hot water bath and give them to me so I could pipette the ingredients in. She would then put the samples back in the hot water bath to continue heating them. While she was working with radioactive materials, I prepared two Agarose gels. I measured out the Agarose, added the TAE or TBE buffer, dissolved the Agarose for 2 minutes and 15 seconds, let the mixture cool, and then poured the gel liquid into the apparatus to let it settle. This time, Dr. Bermek loaded the samples into the gel because some of them were radioactive and required special precautions. I didn’t trust myself enough to load after we had spent about 3 hours preparing the samples.
While the gel was running we prepared grids for the EM that we would use tomorrow. When I first used grids last week, they were already prepared so I was glad I got to see the full process today. There was a special solution we poured over the mesh where our grids would be housed. Once the mesh was completely submerged in the liquid, we carefully placed the grids one by one on the mesh making sure they stuck. I got to help Dr. Bermek with this step, and I really liked the precise nature of it. Then came the coolest part: adding the Parlodion. Parlodion is used to coat the gel grids so they can be used in the EM. The coolest thing was when we added a few drops of it to the solution that the mesh was in, it created a rainbow colored layer over the grids. I was really fascinated.
By the time we had finished the UL8 experiment and set up the grids, they day was just about over. Even though we may not get the results that weren’t likely to happen, I’m excited to see what the gel image and EM show us tomorrow on my last day working with Dr. Bermek.
The gel that contains radioactive material getting run behind a protective shield.
Getting ready to add the Parlodion.
The cool rainbow effect of the Parlodion.
The Agarose gel I set up.
Our designed experiment plans for the day.
