Pglo Transformation Essay

Words: 1840
Pages: 8

Connor Lauffenburger
pGlo Transformation Lab Report
I Introduction
The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies
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The pellet was re-suspended by rapidly pipetting the solution up and down. Once again using a new pipette, a single drop of lysozyme was added to the solution and the tube was capped and mixed. Before placing the tube in the freezer until the next lab period, the pellet was viewed under the UV light.
After thawing the tube out by hand, the tube was placed in the centrifuge for 10 minutes at maximum speed. While waiting on the centrifuge, the chromatography column was shaken to re-suspend the beads. Next, the cap and bottom of the chromatography column was removed and the liquid buffer was drained. After the buffer completely drained, 2 milliliters of Equilibration Buffer was added to the top of the column, and then drained until there was only 1 milliliter using a pipette. The top and bottom of the column were sealed and the column was placed at room temperature until the next lab period. After the tube was done with centrifugation, it was immediately placed under the UV light and examined for a pellet at the bottom. Notes were taken on the color of the pellet and liquid and, using a sterile pipette, 250 microliters of the supernatant was transferred into a new microtube. Next, 250 μl of Binding Buffer was added to the microtube containing the supernatant and the tube was refrigerated until the following lab period.
After labeling 4 collection tubes 1, 2, 3, and waste, the top and bottom of the chromatography column were removed and