Recombinant Dna Paper

Recombinant Lab #1

Abstract

Recombinant DNA Lab is focused on creating a genomic library and cloning the luxAB gene from Vibrio fischeri. There are two ways to creating a genomic library. Approach one is to clone from a library. Approach one is great for genes that are not well known by creating a library and screening the colonies for the gene or protein. Throughout the lab we will be able to do both the approaches. In lab number one we start off by purifying the genomic DNA from Vibrio fischeri. In order to isolate the DNA from bacteria we grew up enough of the bacteria by placing a small amount of the bacteria in a flask with appropriate media or innoculation. We then incubated the broth with the bacteria at an appropriate temperature until the bacteria has reached a high concentration. We then centrifuged the broth to collect the bacteria in order to proceed to the next step of lysing the DNA. Second part of lab one was purification of plasmid DNA using Quiagen miniprep kit. Using plasmid pGEM-3Zf(+) as the vector to create the library, we isolated the pGEM from host bacterial cells and stored it for later use in creating the library.

In first part of the lab I observed we used many buffers, and enzymes such as Lysozyme, Proteinase K, SDS, Phenol, and TES to help break down and destabilize the cell in order to get to our DNA. At one point we used phenol to denature proteins, and in step 9 of lab 2 we noticed that during the denaturing of proteins by phenol we were able to observe the separation of the aqueous and organic layers. We were also able to notice the great fragility of DNA, and we learned to be careful with our samples.

The purpose of lab three was to continue the isolation of Vibrio fischeri DNA that was began from the last lab. In lab three we specifically finished the purification and quantitated both of our genomic and plasmid DNA. Before creation of our library, we determined if we were successful at our DNA purification. In order...