《gfp purification - embrace challenge：绿色荧光蛋白纯化-接受挑战》由会员分享，可在线阅读，更多相关《gfp purification - embrace challenge：绿色荧光蛋白纯化-接受挑战（2页珍藏版）》请在金锄头文库上搜索。
1、GFP Purification Questions to answer in lab notebookAnswer questions in your lab notebook. If you do not copy the question, then answer in a complete sentence. Lesson 1 Finding the Green Fluorescent Molecule- answers stamped on May 2Genetic Transformation ReviewIn Bio-Rad Kit 1, you performed a gene
2、tic transformation of E. coli bacterial cells. Theresults of this procedure were colonies of cells that fluoresced when exposed to ultravioletlight. This is not a normal phenotype (characteristic) for E.coli. You were then asked to figureout a way to determine which molecule was becoming fluorescent
3、 under UV light. Afterdetermining that the pGLO plasmid DNA was not responsible for the fluorescence under theUV light, you concluded that it was not the plasmid DNA that was fluorescing in response tothe ultraviolet light within the cells. This then led to the next hypothesis that if it is not the
4、DNAfluorescing when exposed to the UV light, then it must be a protein that the new DNA produceswithin the cells.1. Proteins.a. What is a protein?b. List three examples of proteins found in your body.c. Explain the relationship between genes and proteins.2. Using your own words, describe cloning.3.
5、Describe how the bacterial cloned cells on your LB/amp plate differ from the cells onyour LB/amp/ara plate. Design an experiment to show that both plates of clonedcells behave similarly and do contain the same DNA?Lesson 2 Capturing colonies and growing in liquid media- answers stamped on May 2Revie
6、w Questions1. What is a bacterial colony?2. Why did you pick one green colony and one white colony from your agar plate(s)? Whydo you think you picked one of each color? What could this prove?3. How are these items helpful in this cloning experiment?a. ultraviolet (UV) light -b. incubator -c. shakin
7、g incubator -4. Explain how placing cloned cells in nutrient broth to multiply relates to your overall goalof purifying the fluorescent protein. In other words, why are we inoculating liquid media with the bacteria in the first place?Lesson 3 Eeks, my cultures are green! - answers stamped on May 9Re
8、view Questions1. You have used a bacterium to propagate a gene that produces a green fluorescent protein.Identify the function of these items you need in Lesson 3.a. Centrifuge -b. Lysozyme -c. Freezer -2. Do you expect both liquid cultures to fluoresce green? Why/why not?3. Why did you discard the
9、supernatant in this part of the protein purification procedure?4. Can you explain why the bacterial cells outer membrane ruptures when the cells arefrozen. What happens to an unopened soft drink when it freezes?5. What was the purpose of rupturing or lysing the bacteria?Lesson 4 Centrifugation, keep
10、 or toss the pellet? Why? - answers stamped on May 9Review Questions1. What color was the pellet in this step of the experiment? What color was the supernatant?What does this tell you?2. Why did you discard the pellet in this part of the protein purification procedure?3. Briefly describe hydrophobic
11、 interaction chromatography and identify its purpose in thislab.Lesson 5 Watch the glow move through the column - prediction stamped on May 9 (prediction stamped, not the direct observation) Answers to the questions based on observations are due on May 14Review Questions1. List your predictions and
12、observations for the sample and what happens to the samplewhen the following buffers are added to the HIC column. (Make a copy of this table in your lab notebook.)Collection Tube NumberPredictionObservationsUnder UV Light(column and collection tube)Tube 1Sample in Binding BufferTube 2Sample with Was
13、h BufferTube 3Sample with Elution Buffer2. Using the data table above, compare how your predictions matched up with your observationsfor each buffer.a. Binding Buffer-b. Wash Buffer-c. Elution Buffer-3. Based on your results, explain the roles or functions of these buffers. Hint: how does thename of
14、 the buffer relate to its function.a. Equilibration Buffer- b. Binding Buffer- c. Wash Buffer- d. TE (Elution) Buffer-4. Which buffers have the highest salt content and which have the least? How can you tell?5. Were you successful in isolating and purifying GFP from the cloned bacterial cells?Identify the evidence you have to support your answer.
