Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

CHAPTER 5 100 ml CULTURE PROTOCOL Protein Expression and Purification Series Chapter 5: Culturing, Expression, Lysis and SDS- PAGE Analysis for 100 ml Cultures for Chromatography Instrumentation 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 Using the rehydrated BL21(DE3) E. coli containing pHDFR plasmid 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 (Figure 5.1a). 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 (Figure 5.1b) for a total of about 10 times. 3. Rotate the plate again and repeat streaking (Figure 5.1c). Rotate the plate for the final time and make the final streak (Figure 4. 5.1d). When you are finished streaking the plate, cover it immediately to avoid contamination. Label your plate with your initials. 118 Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification
Protein Expression and Purification Series Figure 5.1. Streaking a plate to generate single colonies. 5. Place the plate upside down inside the incubator overnight at 37°C. Once colonies have grown to 1 mm in diameter they may be stored in a sealed bag upside down at 4ºC until ready to use for up to one month. 6. E. coli forms off-white colonies that are uniformly circular with smooth edges. Avoid using plates with contaminant colonies. Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification 119 CHAPTER 5 100 ml CULTURE PROTOCOL
Page 1:
Biotechnology Explorer TM Protein E
Page 4 and 5:
Protein Expression and Purification
Page 6 and 7:
Page 8 and 9:
Page 10 and 11:
Page 12 and 13:
Page 14 and 15:
Page 16 and 17:
Page 18 and 19:
Page 20 and 21:
Page 22 and 23:
Page 24 and 25:
Page 26 and 27:
Page 28 and 29:
Page 30 and 31:
Page 32 and 33:
Page 34 and 35:
CHAPTER 1 BACKGROUND Protein Expres
Page 36 and 37:
Page 38 and 39:
Page 40 and 41:
Page 42 and 43:
Page 44 and 45:
Page 46 and 47:
Page 48 and 49:
Page 50 and 51:
CHAPTER 2 FOCUS QUESTIONS Protein E
Page 52 and 53:
Page 54 and 55:
Page 56 and 57:
CHAPTER 3A ADVANCE PREP CENTRIFUGAT
Page 58 and 59:
Page 60 and 61:
Page 62 and 63:
Page 64 and 65:
Page 66 and 67:
Page 68 and 69: CHAPTER 3A ADVANCE PREP CENTRIFUGAT
Page 70 and 71: CHAPTER 3A ADVANCE PREP CENTRIFUGAT
Page 72 and 73: CHAPTER 3B ADVANCE PREP INSTRUMENTA
Page 92 and 93: CHAPTER 4 11 ml CULTURE PROTOCOL Pr
Page 120 and 121: CHAPTER 5 100 ml CULTURE PROTOCOL P
Page 134 and 135: CHAPTER 6 HANDPACKING COLUMNs Prote
Page 140 and 141: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 168 and 169:
CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
APPENDIX A TIPS AND HELPFUL HINTS P
Page 212 and 213:
APPENDIX A TIPS AND HELPFUL HINTS P
Page 214 and 215:
APPENDIX B RESULTS ANALYSIS Protein
Page 216 and 217:
APPENDIX B RESULTS ANALYSIS Protein
Page 218 and 219:
APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221:
APPENDIX D SETTING UP THE SMARTSPEC
Page 222 and 223:
APPENDIX D SETTING UP THE SMARTSPEC
Page 224 and 225:
APPENDIX E MINI-PROTEAN GEL BOX SET
Page 226 and 227:
APPENDIX F BIOLOGIC LP SYSTEM SETUP
Page 228 and 229:
APPENDIX F BIOLOGIC LP SYSTEM SETUP
Page 230 and 231:
APPENDIX G BIOMANUFACTURING Protein
Page 232 and 233:
APPENDIX G BIOMANUFACTURING Protein
Page 234 and 235:
APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 236 and 237:
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
APPENDIX I GLOSSARY Protein Express
Page 244 and 245:
APPENDIX I GLOSSARY Protein Express
Page 246 and 247:
APPENDIX J REFERENCES, LEGAL NOTES
Page 250:
Life Science Group Bulletin 1665067
show all

Protein Expression and Purification Series - Bio-Rad

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?