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Name: _____________________________________________ Date: ____________ Per: ____
Protein Purification: Green Fluorescent Protein
Student Lab Report
Instructions: Read all sections of the lab report carefully and use full sentences to
explain all answers. Use your lab report rubric to complete all requirements for
full credit.
Background: Define the following genetic engineering vocabulary terms. (10 points)
1. Centrifuge
2. Plasmid
3. Green Fluorescent Protein
4. Supernatant
5. Buffer
6. Micro-vial
7. Cell Culture
8. Pellet
9. Precipitate
10. Resuspension
Class Materials
100 ml E.Coli Cell Culture in Media (BL21-GFP overnight culture LB + Amp Media)
UV Light
Centrifuge
Lab Group Materials
1 ml Bacteria Culture
1 ml Buffer A: High Salt (H2O, 1M NaCl, 10% Glycerol)
5 ml Buffer B: Low Salt (H2O, 0.1M NaCl, 10% Glycerol)
Micro-centrifuge tubes
15 ml test tubes
2 ml Cell Lysis Solution (25% liquid detergent in H2O)
Micropipette: 1000 microliter (µl)
Permanent Marker
Test tube rack
Waste Container (cup/beaker)
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Procedure: Follow steps carefully in each of the sections below. (5 points)
Part 1:
1. Before getting into lab groups, watch for materials demonstrations and initial cell culture
introduction as a whole class.
2. The 100 ml cell culture will be placed under UV light to look for the presence of GFP.
Watch carefully and describe initial observations of 100 ml cell culture in data table.
Part 2:
3. In assigned lab groups, obtain all needed materials.
4. Label one micro-centrifuge tube “cells” with your lab group number on the top and side
with your permanent marker.
5. Obtain 1 ml of initial cell culture in “cells” micro-centrifuge tube.
6. Spin down your “cells” tube in centrifuge for 5 minutes to create cell pellet.
7. Remove all liquid from your tube by pipetting and disposing in waste
container. Only your cell pellet should remain at the bottom of the tube.
8. Add 1000 µl of Buffer A to your “cells” tube to resuspend your cell pellet.
9. Use your micropipette to transfer all resuspended cells into 15 ml test tube.
10. Slowly add 2 ml of cell lysis solution to test tube. Place test tube in rack and
wait a few minutes to allow all cells to break open. Your GFP should be free
in solution.
11. Use your micropipette to transfer 1000 µl from your test tube into a new
micro-centrifuge tube. Label your new micro-centrifuge tube “First GFP” with
lab group number.
12. Spin down your GFP tube in centrifuge for 5 minutes to remove cell debris.
13. Place First GFP tube under UV light and record observations in Data Table
under First GFP purification. Describe both the supernatant and cell debris.
Where is the green fluorescence located?
Part 3:
14. Transfer supernatant from GFP tube to new 15 ml test tube.
15. Add 5 ml of Buffer B and mix well. Let tube sit for 15 minutes to allow GFP
protein to precipitate out of solution.
16. Use micropipette to draw up 1000 µl of solution with precipitate mixed in.
Place into new micro-centrifuge tube and label “Second GFP” with group
number.
17. Spin down your Second GFP tube in centrifuge for 5 minutes to create
purified GFP protein pellet.
18. Place Second GFP tube under UV light and record observations in Data
Table under Second GFP purification. Describe both the supernatant and cell
debris. Where is the green fluorescence located?
19. Place your “First GFP” and “Second GFP” tubes into rack at front of room for
whole class viewing at the end of the period.
20. Clean up all materials as instructed. Complete all portions of lab report following
instructions for full credit.
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Data Tables (5 points)
Day/Time
Bacteria Description: Use qualitative and quantitative observations, any
available data, make best possible descriptions.
Initial 100 ml
Cell Cutlure
First GFP
purification
Second GFP
purification
Conclusion (10 points)
Explain all answers with full sentences using specific data from the experiment and
your knowledge of genetic transformation and protein purification.
1. The initial cell culture contained E.Coli bacteria that were genetically transformed to contain
the GFP gene. Why was only a low level of GFP visible in the initial cell culture?
2. Why purpose did the cell lysis solution serve for this procedure? What was its result?
3. Was GFP soluble or insoluble in Buffer A? How do you know?
4. Was GFP soluble or insoluble in Buffer B? How do you know?
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5. Compare the Initial Cell Culture observations to your First GFP and Second GFP
observations. Where was the most GFP observed? Explain why.
6. Explain how this type of protein purification procedure could be used in a real world
application of genetic transformation. What other types of proteins could be produced and
purified to benefit humans?
7. Use the following terms to describe the overall process of genetic transformation in bacteria
cells which made this lab procedure possible: bacteria cell, plasmid, gene, transformation,
DNA, RNA, Protein. You may draw picture or a flow chart to help also. (4 points)
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