These past two weeks have involved a lot more bacteria and mini/midi preps for me. I have been mainly focused on finishing the molecular cloning project, and we are almost done with the last two DNA constructs, hSyn-hM3D-2a-smRuby-V5-pA and hSyn-ReaChr-2a-smRubyMyc-pA. To wrap up the molecular cloning project, I have been spent a good chunk of time with my good friend bacteria (and its wondrous scent). In the last two weeks, I have performed four bacterial transformations, which is the process by which competent bacterial cells take up DNA molecules and replicate the foreign DNA along with their own DNA. For these bacterial transformations, I spread the bacteria onto an LB agar plate treated with either ampicillin or kanamycin (depending on what plasmid we inserted into the bacteria) so that only colonies of bacteria with our plasmid will grow. Then, after allowing the colonies to grow overnight in an incubator, I will harvest ten of the colonies using micropipette tips, eject each tip into a tube with 5 mL of LB broth and 5 µL of ampicillin/kanamycin, incubate the tubes on a shaker overnight and then prepare mini preps.
Indeed, I also have performed a myriad of mini and midi preps in the past two weeks. As I mentioned in my previous post, mini and midi preps are used to isolate the plasmid DNA from the bacteria. Unfortunately, the midi prep is probably my least favorite step of the cloning process. While the mini preps take approximately an hour and a half to finish, the midi preps require almost the entire day to complete. Spending a whole day in a whirlwind of falcon tubes, eppendorf tubes, erlenmeyer flasks, filters, and columns is, sadly, not the most exciting thing ever. To make matters worse, during my first midi prep, one of my falcon tubes shattered in the large centrifuge, causing an explosion of bacterial solution and its pungent aroma. Luckily, nothing else besides that tube broke. In summary, my experience with midi preps so far has been quite hectic.
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| When you can't see the DNA pellet |
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| The midi prep: featuring my cluttered desk |
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| More bacterial colonies! |
Addie, a member of the lab next door, was kind enough to explain to me the next step, a viral transfection, after the engineering of the DNA construct. A viral transfection is the process of introducing purified nucleic acids into eukaryotic cells (in our case the neurons of the mice brains). In order, the steps of a transfection include the creation of the recombinant virus carrying the transgene (gene or genetic material that has been transferred from one organism to another), amplification of the recombinant viral particles in a packaging cell line, purification and titration of the amplified viral particles, and infection of the cells of interest. While Felicia and I were responsible for the creation of the recombinant virus, Addie will be performing the final three steps of the transfection process. Addie also showed me how to prepare qubit samples to analyze under a qubit fluorometer, which is used to quantify DNA, RNA or protein.
The molecular cloning process includes some periods of time of just waiting. During this waiting time, I have continued to cut slices of the mouse brains using the vibratome, mount and cover slip these slices, and observe and image the slices under the episcope.
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| The brain slices |
During these past two weeks, I have also had the opportunity to interact with Dr. Fuccillo frequently. Despite his busy schedule, he has made time to help me with the cloning project, and he was actually the one to teach me how to do a midi prep. Meanwhile, I have become very close with Felicia and Kyuhyun. Even though there is a pretty significant age gap between the three of us, we all treat each other as equals and friends. Like Nick, I definitely love the social aspect of the lab just as much as the scientific aspect. It is really wonderful that everyone in my lab is both dedicated to their work and also amiable and sociable to each other. I can't believe I only have two more weeks left! I am definitely going to miss being in the lab. I hope everyone else is having a great time at their lab!
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| When it's too hot outside: featuring Felicia and Kyuhyun |
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| Being super productive |
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| Absorbing all the molecular cloning knowledge |
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