Protein Expression and Purification Series - Bio-Rad

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CHAPTER 3A ADVANCE PREP CENTRIFUGATION PROCES Protein Expression and Purification Series Chapter 3A: Advance Preparation for Centrifugation Purification Protocols Advance Preparation for Cell Culture and Induction Portions of the Protein Expression and Purification Series In the Cell Culture activities of the Protein Expression and Purification Series, students will grow a small culture of BL21(DE3) E. coli containing pDHFR starting from a single colony from a streaked plate. The initial culture will be subcultured to log phase and then induced to express GST-DHFR-His. Cell Culture Laboratory Checklist Components from Protein Expression and Purification Series Where Provided (✔) Petri dishes Growth and Expression Module ❒ LB nutrient agar Growth and Expression Module ❒ Ampicillin Growth and Expression Module ❒ LB broth capsules Growth and Expression Module ❒ E. coli BL21(DE3) with pDHFR, lyophilized Growth and Expression Module ❒ Sterile 50 ml conical tubes Growth and Expression Module ❒ Sterile inoculating loops Growth and Expression Module ❒ IPTG Growth and Expression Module ❒ Sterile water Growth and Expression Module ❒ 20% sterile glucose Growth and Expression Module ❒ Microcentrifuge tubes, 2 ml Growth and Expression Module ❒ Laemmli sample buffer SDS-PAGE Electrophoresis Module ❒ Screwcap microcentrifuge tubes, 1.5 ml Growth and Expression Module ❒ Required Accessories (Not Provided) Quantity (✔) Incubator set to 37°C 1 ❒ Autoclave, microwave or hot plate and stir bar 1 ❒ Erlenmeyer flask, 500 ml or larger 1 ❒ Tube roller, shaking water bath or shaking incubator set to 37°C 1 ❒ Microcentrifuge with variable speed setting >16,000 x g 1 ❒ 2–20 µl adjustable-volume micropipets and tips 12 ❒ 20–200 µl adjustable-volume micropipets and tips 12 ❒ 100–1,000 µl adjustable-volume micropipets and tips 12 ❒ Pipet controller or bulb 12 ❒ 10 ml sterile serological pipets 5–10 ❒ Marking pens 12 ❒ Spectrophotometer 1 ❒ Semi-microvolume cuvettes 48 ❒ Water bath or heat block set to 95°C 1 ❒ 56 Chapter 3A: Advance Preparation for Centrifugation Protocols
Protein Expression and Purification Series Tasks to Perform Prior to the Lab Note: Several reagents are used in different parts of the advance preparation as well as for different laboratory activities. Do not discard excess reagents used in preparation and store them at the appropriate conditions. Streaking starter plates to produce single bacterial colonies on agar plates 1. Preparation of ampicillin: With a sterile pipet, add 600 µl of sterile water to each vial containing 30 mg ampicillin to rehydrate the antibiotic, making a 50 mg/ml (500x) solution. Note: Ampicillin is used to make LB/amp agar plates as well as LB/amp broth, so do not discard any unused ampicillin solution. The rehydrated ampicillin should be stored at –20°C and is good for one year. 2. Preparation of LB/amp agar plates: At least two days prior to cell culturing, prepare one LB/amp agar plate per team to be used to inoculate E. coli starter cultures. Note: Only prepare 200 ml of molten LB agar reagent (enough for 20 plates) to which 200 µl of 50 mg/ml ampicillin will be added to make LB/amp agar plates. Retain excess 50 mg/ml ampicillin at –20°C for use in preparation of LB/amp broth. This protocol is used to prepare solid LB/amp agar media to prepare plates for the growth of starter colonies of BL21(DE3) E. coli containing the pDHFR plasmid. Each student group will require one LB/ amp agar plate. Agar plates should be prepared at least two days before required, left out at room temperature for two days and then refrigerated until they are to be used. Two days on the bench allows the agar to dry out and more readily take up the rehydrated E. coli solution. Figure 3A.1. Preparation of LB/amp agar. A. To prepare 20 LB/amp plates, prepare 200 ml of LB agar. Add 8 g of LB nutrient agar to 200 ml of distilled water in a 500 ml or larger Erlenmeyer flask and cover the flask opening. Swirl the flask to dissolve the agar, or add a magnetic stir bar to the flask and stir on a stir plate. A stir bar will also aid in mixing the solution once sterilization is complete. Autoclave the LB agar on the wet cycle for 30 minutes. Once the autoclave cycle is complete, check the solution to ensure that the agar is completely dissolved. Note: Be sure to wear appropriate protective equipment and be careful to allow the flask to cool slightly before swirling so that the hot medium does not boil over onto your hand. If no autoclave is available, heat the agar solution to boiling in a microwave or on a hot plate. Repeat the heating and swirling process about three times until all the agar is dissolved (no more clear specks are noticeable). Use a lower power setting on the microwave to reduce evaporation and boilover. Allow the LB agar to cool so that the outside of the flask is just comfortable to hold (around 55°C). A water Chapter 3A: Advance Preparation for Centrifugation Protocols 57 CHAPTER 3A ADVANCE PREP CENTRIFUGATION PROCES
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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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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