bevtest 11.45.23 PM - Instruction Manual Guide to...

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Instruction Manual Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques
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INSIDE FRONT COVER
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i 1. Introduction Overview of Baculovirology. ........................................................................................................................................... 1 Baculoviruses as Expression Vectors . ........................................................................................................................... 1 Advantages of BEVS Technology. .................................................................................................................................. 2 Generating a Recombinant Virus by Homologous Recombination. ................................................................................ 3 Generating a Recombinant Virus by Site-Specific Transposition. ................................................................................... 3 Insect Cell Culture Techniques. ...................................................................................................................................... 4 2. Protocols for Culturing Host Cells ............................................................................................................................. 6 Protocol 1: Subculturing Monolayer Cultures. ........................................................................................................... 6 Protocol 2: Adapting Monolayer Cells to Suspension Culture. ................................................................................. 6 Protocol 3: Maintaining Suspension Cultures . .......................................................................................................... 7 Protocol 4: Adapting Cultures to Serum-Free Medium. ............................................................................................ 8 Protocol 5: Preparing a Master Cell Seed Stock . ..................................................................................................... 8 3. Protocols for Generating a Recombinant Baculovirus .......................................................................................... 10 Protocol 6: Isolation of Bacmid DNA for B AC - TO -B AC ® Baculovirus Expression System with the C ONCERT High Purity Plasmid Purification System. ............................................................... 10 Protocol 7: Cationic Liposome-Mediated Transfection Using C ELL FECTIN Reagent . ............................................ 11 Protocol 8: Virus Plaque Assay . .............................................................................................................................. 4. Protocols for Purifying and Producing Recombinant AcNPV and Protein ......................................................... 14 Protocol 9: Plaque Purification of Recombinant Viral Clones. ................................................................................ 14 Protocol 10: Amplifying the Virus Stock . ................................................................................................................... 14 Protocol 11: Identifying Plaques by Neutral Red Staining . ....................................................................................... 14 Protocol 12: Optimizing Virus Stock Production . ...................................................................................................... 15 Protocol 13: Harvesting the Virus . ............................................................................................................................ 15 Protocol 14: Concentrating the Virus . ....................................................................................................................... 15 Protocol 15: Storing the Virus . .................................................................................................................................. 16 Protocol 16: Optimizing Heterologous Protein Production. ....................................................................................... 16 5. Purifying Recombinant Proteins ............................................................................................................................... 17 Purifying Secreted Proteins. .......................................................................................................................................... 17 Purifying Intracellular Proteins. ...................................................................................................................................... 18 6. References ................................................................................................................................................................ 21 7. Related Products ........................................................................................................................................................ 22 Appendix A: Applications Data for Insect Cell Lines Grown in Serum-Free Media ....................................................... 23 Figures: 1 In vivo baculovirus infection and replication. ....................................................................................................................... 2 2 Generating a recombinant baculovirus by homologous recombination. .............................................................................. 3 3 Generation of recombinant baculoviruses and gene expression with the B AC - TO -B AC expression system . ....................... 4 Table of Contents
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ii Tables: 1 Insect cell lines commonly used in BEVS applications . ...................................................................................................... 4 2 Insect cell culture media commonly used in BEVS applications. ........................................................................................ 4 3 Effects of serum on cell cultures. ......................................................................................................................................... 5 4 Useful medium volumes . ..................................................................................................................................................... 7 5 Troubleshooting virus plaque assays . ............................................................................................................................... 13 6 Recommended maximum infection densities for the production of rAcNPV or recombinant products. ............................ 14 7 Maximum cell densities in small-scale suspension culture . .............................................................................................. 22 8 β -galactosidase expression in small-scale suspension culture . ........................................................................................ 23 9 Pilot-scale recombinant protein expression in cells cultured. ............................................................................................ 23 10 rAcNPV titers in small-scale suspension culture . .............................................................................................................. 23 B AC-TO-BAC ® , C ELLFECTIN ® , DH10B AC , pF ASTBAC 1, E XPRESS-FIVE ® , C ONCERT ,T ECH-LINE SM , and the Life Technologies logo are marks of Life Technologies, Inc. The B AC-TO-BAC Baculovirus Expression System is sold under patent license for research purposes only, and no license for commercial use is included.
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bevtest 11.45.23 PM - Instruction Manual Guide to...

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