March 30, 2021
Catie Fleischer and Carter Beck
Introduction:
We are part of the Cancer and Immunity Stream with Dr. Land at Minnesota State University, Mankato. Our research in this stream is focused on how miRNAs (replicated RNA fragments that may interfere with the expression of mRNA genes) interact with APOBEC3A and APOBEC3B. The miRNAs we are researching are hsa-mir-29a-3p and hsa-mir-655-5p, which have been found to be involved in a variety of different cancers.
Specific Content:
Over the past two weeks we have been working on a bacterial transformation and plasmid purification with E. coli bacterial cells to
collect the ampicillin resistant plasmid DNA that we will use in a later part of our research.
For the bacterial transformation, we added the E. coli to two tubes and added the ampicillin plasmid only to one tube to act as a control for this process. We then heat shocked the cells by incubating the E. coli on ice for 20 minutes, then placing the cells in a 42℃ heat bath for exactly 30 seconds, and then placing the cells in a 37℃ incubator for 60 minutes. This put the cells in an unstable state so that they would pick up the ampicillin resistant plasmid that we added. After the 60 minutes were over we used two methods to deliver the cells to the growth plates: streaking and spreading. The growth plates we used had an ampicillin on them, so we could expect the cells with the plasmid added to grow and the cells without the plasmid not to grow, which is what happened.
Since our cell growth was as we expected we were then able to begin isolating and purifying our samples. For this procedure, we added a few colonies from the growth plate into culture tubes, each containing a liquid medium and let those grow overnight. The next day we prepared the plasmid DNA. We did this by removing any substances we didn’t want such as proteins by centrifuging the solution after we added the specified substance from the kit we used. Then we lysed the cells by adding a substance from the kit and letting the tube sit for 2 minutes to only allow the plasmid to escape the cell and not the chromosomal DNA. Then once we had finished purifying the plasmid DNA we checked to see the DNA concentration with the nanodrop, which showed that we had a good concentration of DNA, proteins, and salt.
Reflection:
Overall, this process went smoothly and we learned a lot from the process and everything we have done in the lab thus far. It’s always nice to have things work the first time because they don’t always, but that’s what makes it even more rewarding when things go right. We are looking forward to what our research will bring as the semester continues.
