My mentor is currently investigating DNA repair and Mitotic Recombination in Yeast. My mentor and I are in the process of deleting a gene coding for a specific enzyme in Yeast. The ultimate goal is to observe the effect this deletion presents to DNA repair and Mitotic Recombination in yeast. This enzyme that my mentor and I are currently in the process of deleting has been observed to regulate certain aspects of mitotic recombination.
To delete this enzyme, a specific set of procedures are followed. This set of procedures involves designing new primers to be amplified through PCR. These primers amplify the targeted region coding for the enzyme, and include a multi-cloning site that allows a drug-resistant gene (with a homologous multi-cloning site) to knockout the targeted enzyme gene. We check PCR products by running gels; we transform amplified DNA to yeast, replica plate the yeast onto YPD + drug plates, purify onto YPD + drug plates (to screen for individual colonies that are drug resistant and may have knocked out the specified gene coding for the targeted enzyme), prep the new DNA, and run gels to verify if the targeted gene (coding for the enzyme) is actually deleted and if the drug-resistant gene is in its place.
We are currently on our second attempt at deleting this targeted gene that codes for this specific enzyme. The first attempt came out negative because the drug did not knockout the targeted gene; instead the drug (gene) lied down at another location in the yeast genome. Our second attempt includes some modifications in the screening process.
I am really enjoying working in lab. I’m learning a lot of new techniques dealing with DNA, and I am beginning to feel more comfortable working in a lab environment. I hope to continue working in this lab and working on this project in the future.