Protein Expression and Purification Series - Bio-Rad

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CHAPTER 3B ADVANCE PREP INSTRUMENTATION PROCES Protein Expression and Purification Series kinetics mode can be programmed and how to set the wavelength at 340 nm. If there is no kinetics mode, readings at 340 nm can be taken manually every 15 seconds for 150 seconds and written down. Note: UV compatible cuvettes (trUView or quartz) that can read 1 ml samples must be used for this activity. 5. Aliquot 1 ml of 1x PBS into a 2 ml microcentrifuge tube for each workstation. 6. Set up student workstations according to the student workstation list below. Student Workstations Each student team requires the following items to analyze enzymatic activity of their purified, desalted GST-DHFR-His protein sample: Material Needed for Each Workstation Quantity Purified, desalted eluate protein sample 15 µl 2–20 µl adjustable-volume micropipet and tips 1 100–1,000 adjustable-volume micropipet and tips 1 1x PBS 1 ml Marking pen 1 NADPH cofactor 8 µl DHF substrate 10 µl trUView disposable cuvettes (or UV compatible cuvettes) 1–3 Parafilm 1 square Common Workstation Quantity UV spectrophotometer capable of three decimal place accuracy 1–2 90 Chapter 3B: Advance Preparation for Chromatography Instrumentation Protocols
Protein Expression and Purification Series Chapter 4: Culturing, Expression, Lysis and SDS-PAGE Analysis for 11 ml Cultures for Centrifugation Purification Process Cell Culture and Induction Student Workstations Each student team requires the following items to streak a starter plate to produce single bacterial colonies: Material Needed for Each Workstation Quantity LB/amp plate 1 Sterile inoculating loop 1 2–20 µl adjustable-volume micropipet and tips 1 Marking pen 1 Common Workstation Quantity Rehydrated BL21(DE3) E. coli containing pDHFR 1 Incubator set to 37°C 1 Each lab team will streak their own starter plate as a source of cells for culture for protein production. LB/amp plates should be streaked for single colonies and incubated at 37°C for 16–24 hours before the initial culture activity is planned. Student Protocol: Streaking starter plates to produce single bacterial colonies on agar plates Using the rehydrated BL21(DE3) E. coli containing pDHFR at the Common Workstation to streak one plate. The purpose of streaking is to generate single colonies from a concentrated suspension of bacteria. A minute amount of the bacterial suspension goes a long way. Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours. There are millions of individual bacteria in a single 1 mm bacterial colony. 1. Pipet 10 µl of reconstituted E. coli using a sterile pipet tip onto an LB/amp plate. Use a sterile loop to perform the streaking. Streaking takes place sequentially in four quadrants. The first streak is to just spread the cells out a little. Go back and forth with the loop about a dozen times in one quadrant as shown in Figure 4.1a. In subsequent quadrants the cells become more and more dilute, increasing the likelihood of producing single colonies. 2. For subsequent streaks, the goal is to use as much of the surface area of the plate as possible. Rotate the plate approximately 45 degrees (so that the streaking motion is comfortable for your hand) and start the second streak. Do not dip into the rehydrated bacteria a second time. Go into the previous streak about two times and then back and forth as shown in Figure 4.1b for a total of about 10 times. 3. Rotate the plate again and repeat streaking (Figure 4.1c). Rotate the plate for the final time and make the final streak (Figure 4. 4.1d). When you are finished streaking the plate, cover it immediately to avoid contamination. Label the plate with your initials. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 91 CHAPTER 4 11 ml CULTURE PROTOCOL
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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CHAPTER 1 BACKGROUND Protein Expres
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX C RCF TO RPM CONVERSTION P
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX E MINI-PROTEAN GEL BOX SET
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX H FOCUS QUESTIONS INSTRUCT
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APPENDIX I GLOSSARY Protein Express
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APPENDIX I GLOSSARY Protein Express
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APPENDIX J REFERENCES, LEGAL NOTES
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Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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