I wrote in my last two blog posts that my molecular cloning project is almost finished, but unfortunately, this week Felicia and I got stuck on the final step of the engineering of the construct hSyn-hM3D-2a-smRuby-V5-pA. In order to create this construct, we need to cut the vector, hSyn-ChiEF-2a-Venus, using the enzymes SalI-HF and XbaI in order to remove the ChiEF-2a-Venus and then later ligate the hSyn fragment with the insert, hm3D-2a-smRuby-V5-pA. We attempted but failed the digest of hSyn-ChiEF-2a-Venus twice using the two aforementioned enzymes, so we are currently troubleshooting to find the root cause of this issue. As of now, we have figured out that the problem is not with the enzymes. We think the problem is either 1.) the hSyn-ChiEF-2a-Venus DNA is contaminated or 2.) we are not digesting hSyn-ChiEF-2a-Venus DNA for enough time. Hopefully, we will be able to resolve the issue and complete the construct before I leave.
This past Thursday, Felicia allowed me to perform my first perfusion under her supervision. As I mentioned in a previous post, a perfusion is the process by which we kill the mouse, inject paraformaldehyde (PFA) into the left ventricle of its heart so the PFA perfuses through the mouse's body and drains the mouse's blood, and extract the brain of the mouse to slice and observe it under an episcope. Although I have already shadowed many perfusions, I never realized how difficult they are as the graduate students and postdocs made the perfusion process seem so easy.
Firstly, I found that the mouse's skin is surprisingly thick and difficult to cut. Additionally, I found it difficult to determine if my needle was injecting PFA into the right part of the mouse's heart since the heart is so small. A perfusion is successful if the mouse's brain turns white because all the blood should be drained. When I cut open my mouse's head to view the brain, my mouse's brain was still a little bit pink, indicating that the perfusion was not entirely successful. Extracting the brain from the mouse's skull was the most difficult part of the perfusion for me. The thick layer surrounding the brain is quite difficult to peel off, and I ended up slightly impaling the brain while trying to remove it with tweezers. Even though it wasn't a completely successful perfusion, I am still glad that I was able to be exposed to a process that is very different from the midi preps, bacterial transformations and molecular cloning techniques that I am used to performing. To be honest, I have wanted to do a perfusion myself for a while, and I am very grateful that Felicia allowed me and helped me to perform one.
In addition to the molecular cloning and the perfusion, I have sliced another two brains this week and performed immunohistochemical staining on slices from both of the brains.
 |
| My first perfusion! |
This past Friday, Felicia left for California to spend two weeks at home with her family, so I had to bid her farewell on Friday afternoon. She was kind enough to gift me with a book named
Brilliant Blunders: From Darwin to Einstein, as well as a card thanking me for working with her this summer. We have promised each other that we will stay in contact, but I am definitely going to miss being with her in the lab and singing along to christmas carols together in the middle of summer. She has been an amazing mentor and friend to me in these past six weeks. Perhaps I will be able to return next year to work with or simply visit her again!
 |
| The book |
I still cannot believe that I only have one week left here at the Fuccillo lab. Looking back on the past six weeks, one important lesson I have learned is that instead of being afraid of and beating myself up over my mistakes and failures, it is wiser and more efficient to learn from these blunders and to try my best to solve the dilemmas I run into during my experiments. Even though I walked into the Fuccillo lab telling myself "don't you dare make a mistake", I will walk out of the lab encouraging myself to not fear failure and instead take advantage of and learn from it.
No comments:
Post a Comment