Invitrogen Bac to Bac Expression System

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Product Qualification pFastBac Vectors 1. Each vector is tested for transposition efficiency into bacmid DNA upon transformation into MAX Efficiency ® DH10Bac competent cells following the procedure on page 21. Transposition frequency should be greater than 8% (% white colonies). 2. Restriction analysis with the enzymes below is performed on each vector to confirm its identity. Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel. Vector Restriction Enzyme Expected Fragments (bp) pFastBac 1 BamH I Bgl II EcoR I Hind III Not I Sal I 4776 470, 4306 4776 4776 4776 4776 pFastBac 1-Gus BamH I Bgl II EcoR I Hind III Not I Sal I 6661 2508, 2886 1267, 5394 6661 6661 1886, 4775 pFastBac HT A BamH I Bgl II EcoR I EcoR V Hind III Not I Sal I 4856 1080, 1268, 2508 4856 1263, 3593 4856 4856 4856 pFastBac HT B BamH I Bgl II EcoR I EcoR V Hind III Not I Sal I 4857 470, 4387 4857 404, 1263, 3190 4857 4857 4857 continued on next page 66
Product Qualification, continued pFastBac Vectors, continued Vector Restriction Enzyme Expected Fragments (bp) pFastBac HT C pFastBac HT- CAT pFastBac Dual pFastBac Dual- Gus/CAT BamH I Bgl II EcoR I EcoR V Hind III Not I Sal I BamH I Bgl II EcoR I EcoR V Hind III Sal I BamH I Bgl II EcoR I Hind III Nco I Not I Sal I Acc I Nco I Sal I 4858 470, 1474, 2914 4858 1263, 3595 4858 4858 4858 707, 4793 1268, 1724, 2508 5500 1263, 4237 5500 5500 5238 470, 4768 5238 5238 5238 5238 5238 479, 2028, 5336 852, 6991 7843 Cellfectin ® Reagent Cellfectin ® Reagent is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of COS-7 cells with a luciferase reporter-containing plasmid. continued on next page 67
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
Page 13 and 14:
The Bac-to-Bac ® Baculovirus Expre
Page 15 and 16:
Culturing Insect Cells General Guid
Page 17 and 18:
Generating the Recombinant pFastBac
Page 19 and 20:
Cloning into pFastBac HT A, B, and
Page 21 and 22:
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 49 and 50: Expressing Your Recombinant Protein
Page 51 and 52: Troubleshooting Cloning into the pF
Page 53 and 54: Troubleshooting, continued Generati
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: Purchaser Notification, continued L
Page 77 and 78: References Anderson, D., Harris, R.
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Invitrogen Bac to Bac Expression System

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