Product Qualification, continued MAX Efficiency ® DH10<strong>Bac</strong> Chemically Competent E. coli 1. MAX Efficiency ® DH10<strong>Bac</strong> competent cells are tested for transformation efficiency using the pUC19 control plasmid included in the kit. Transformed cultures are plated on LB plates containing 100 µg/ml ampicillin and the transformation efficiency is calculated. Transformation efficiency should be greater than 1 x 10 8 cfu/µg pUC19 with non-saturating amounts (50 pg) of DNA, and greater than 1 x 10 5 cfu/µg pUC19 with saturating amounts (25 ng) of DNA. 2. DH10<strong>Bac</strong> competent cells are tested for transposition efficiency using 1 ng of the pFast<strong>Bac</strong> 1-Gus plasmid and following the procedure on page 21. Transposition frequency should be greater than 8% (% white colonies). 3. Untransformed cells are tested for the appropriate antibiotic sensitivity and absence of phage contamination. 68
References Anderson, D., Harris, R., Polayes, D., Ciccarone, V., Donahue, R., Gerard, G., and Jessee, J. (1996). Rapid Generation of Recombinant <strong>Bac</strong>uloviruses and <strong>Expression</strong> of Foreign Genes Using the <strong>Bac</strong>-To-<strong>Bac</strong>® <strong>Bac</strong>ulovirus <strong>Expression</strong> <strong>System</strong>. Focus 17, 53-58. Ausubel, F. M., Brent, R., Kings<strong>to</strong>n, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Pro<strong>to</strong>cols in Molecular Biology (New York: Greene Publishing Associates and Wiley- Interscience). Barry, G. F. (1988). A Broad Host-Range Shuttle <strong>System</strong> for Gene Insertion in<strong>to</strong> the Chromosomes of Gram-negative <strong>Bac</strong>teria. Gene 71, 75-84. Carring<strong>to</strong>n, J. C., and Dougherty, W. G. (1988). A Viral Cleavage Site Cassette: Identification of Amino Acid Sequences Required for Tobacco Etch Virus Polyprotein Processing. Proc. Natl. Acad. Sci. USA 85, 3391-3395. Ciccarone, V. C., Polayes, D., and Luckow, V. A. (1997). Generation of Recombinant <strong>Bac</strong>ulovirus DNA in E. coli Using <strong>Bac</strong>ulovirus Shuttle Vec<strong>to</strong>r, Volume 13, U. Reischt, ed. (To<strong>to</strong>wa, NJ: Humana Press Inc.). Cole, C. N., and Stacy, T. P. (1985). Identification of Sequences in the Herpes Simplex Virus Thymidine Kinase Gene Required for Efficient Processing and Polyadenylation. Mol. Cell. Biol. 5, 2104-2113. Deutscher, M. P. (1990) Guide <strong>to</strong> Protein Purification. In Methods in Enzymology, Vol. 182. (J. N. Abelson and M. I. Simon, eds.) Academic Press, San Diego, CA. Dougherty, W. G., Carring<strong>to</strong>n, J. C., Cary, S. M., and Parks, T. D. (1988). Biochemical and Mutational Analysis of a Plant Virus Polyprotein Cleavage Site. EMBO J. 7, 1281-1287. Hanahan, D. (1983). Studies on Transformation of Escherichia coli with Plasmids. J. Mol. Biol. 166, 557-580. Harris, R., and Polayes, D. (1997). A New <strong>Bac</strong>ulovirus <strong>Expression</strong> Vec<strong>to</strong>r for the Simultaneous <strong>Expression</strong> of Two Heterologous Proteins in the Same Insect Cell. Focus 19, 6-8. Janson, J. C., and Ryden, L. (1989). In Protein Purification: Principles, High Resolution Methods, and Applications (New York: VCH Publishers). Kertbundit, S., Greve, H. d., Deboeck, F., Montagu, M. V., and Hernalsteens, J. P. (1991). In vivo Random β-glucuronidase Gene Fusions in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 88, 5212-5216. King, L. A., and Possee, R. D. (1992). The <strong>Bac</strong>ulovirus <strong>Expression</strong> <strong>System</strong>: A Labora<strong>to</strong>ry Guide (New York, NY: Chapman and Hall). Luckow, V. A. (1991). In Recombinant DNA Technology and Applications, A. Prokop, R. K. Bajpai and C. Ho, eds. (New York: McGraw-Hill). continued on next page 69
- Page 1:
Bac-to-Bac ® Baculovirus Expressio
- Page 5 and 6:
Kit Contents and Storage Types of P
- Page 7 and 8:
Accessory Products Introduction The
- Page 9 and 10:
Introduction Overview Introduction
- Page 11 and 12:
The Bac-to-Bac ® Baculovirus Expre
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The Bac-to-Bac ® Baculovirus Expre
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Culturing Insect Cells General Guid
- Page 17 and 18:
Generating the Recombinant pFastBac
- Page 19 and 20:
Cloning into pFastBac HT A, B, and
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Cloning into pFastBac HT A, B, and
- Page 23 and 24:
Cloning into pFastBac Dual Introdu
- Page 25 and 26: Transformation and Analysis Introdu
- Page 27 and 28: Generating the Recombinant Bacmid T
- Page 29 and 30: Transforming DH10Bac E. coli, cont
- Page 31 and 32: Analyzing Recombinant Bacmid DNA by
- Page 33 and 34: Analyzing Recombinant Bacmid DNA by
- Page 35 and 36: Transfecting Insect Cells, continue
- Page 37 and 38: Isolating P1 Viral Stock Introducti
- Page 39 and 40: Amplifying Your Baculoviral Stock I
- Page 41 and 42: Performing a Viral Plaque Assay Int
- Page 43 and 44: Performing a Viral Plaque Assay, co
- Page 45 and 46: Performing a Viral Plaque Assay, co
- Page 47 and 48: Performing a Viral Plaque Assay, co
- Page 49 and 50: Expressing Your Recombinant Protein
- Page 51 and 52: Troubleshooting Cloning into the pF
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- Page 55 and 56: Troubleshooting, continued Isolatin
- Page 57 and 58: Appendix Recipes Antibiotic Stock S
- Page 59 and 60: Isolating Recombinant Bacmid DNA In
- Page 61 and 62: Map and Features of pFastBac 1 pFa
- Page 63 and 64: Map and Features of pFastBac HT pF
- Page 65 and 66: Map and Features of pFastBac Dual
- Page 67 and 68: Map of pFastBac 1-Gus Description
- Page 69 and 70: Map of pFastBac Dual-Gus/CAT Descr
- Page 71 and 72: Technical Service, continued Limite
- Page 73 and 74: Purchaser Notification, continued L
- Page 75: Product Qualification, continued pF
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