Green Fluorescent Protein Lab
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein. …show more content…
This shows that the transformation was not completely effective and only transformed some of the most competent bacterial cells.
Laboratory Procedure for pGREEN Questions
6. What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have taken up the plasmid?? The AMP is what allows us to identify which bacteria have been taken up in the plasmid; that is its job.
7. What does the phenotype of the transformed colonies tell you? It tells us that pGreen is there because it glows under the black light.
8. What one plate would you first inspect to conclude that the transformation occurred successfully? Why? The plate I would first inspect would be the LB/Amp+plasmid because the selective marker is AMPR and is will grow only in an ampicillin media.
9. Transformation efficiency calculations a. Determine the total mass of plasmid used. 10 x .005 = .05 ug b. Calculate the total volume of cell suspension prepared. .510 ul c. Now calculate the total volume of total cell suspension that was spread on the plate. 100/510 = .196 d. Determine the mass of plasmid in the cell suspension spread. .05 x .196 = .0098 e. Determine the number of colonies per ug plasmid DNA. Express your answer in scientific notation. 17/.0098 = 1.1735 x 103
10. What factors might influence transformation efficiency? Explain the effect of each factor you