Invitrogen Bac to Bac Expression System

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Kit Contents and Storage, continued pFastBac Vectors Each catalog number includes a specific pFastBac vector(s) and a corresponding expression control, and are supplied as detailed below. Store at +4°C. Product Catalog no. pFastBac Vector Expression Control Bac-to-Bac ® Baculovirus Expression System 10359-016 pFastBac 1 Supplied: 20 µl at 0.5 µg/µl in TE, pH 8.0 (10 µg total) Bac-to-Bac ® Vector Kit 10360-014 pFastBac 1 Supplied: 20 µl at 0.5 µg/µl in TE, pH 8.0 (10 µg total) Bac-to-Bac ® HT Vector Kit 10584-027 pFastBac HT A pFastBac HT B pFastBac HT C Supplied: 20 µl each at 0.5 µg/µl in TE, pH 8.0 (10 µg total of each vector) pFastBac Dual 10712-024 pFastBac Dual Supplied: 20 µl at 0.5 µg/µl in TE, pH 8.0 (10 µg total) pFastBac 1-Gus Supplied: 20 µl at 0.2 ng/µl in TE, pH 8.0 (4 ng total) pFastBac 1-Gus Supplied: 20 µl at 0.2 ng/µl in TE, pH 8.0 (4 ng total) pFastBac HT-CAT Supplied: 15 µl at 1 ng/µl in TE, pH 8.0 (15 ng total) pFastBac Dual-Gus/CAT Supplied: 20 µl at 0.2 ng/µl in TE, pH 8.0 (4 ng total) MAX Efficiency ® DH10Bac Competent E. coli Reagents MAX Efficiency ® DH10Bac Chemically Competent E. coli are supplied with the Bac-to-Bac ® Baculovirus Expression System only, and include the following items. Transformation efficiency is 1 x 10 8 cfu/µg DNA. Store at –80°C. -- 5 x Item Composition Amount MAX Efficiency ® Chemically 100 µl Competent DH10Bac pUC19 Control DNA 10 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8 100 µl Genotype of DH10Bac F - mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacX74 recA1 endA1 araD139 ∆(ara, leu)7697 galU galK λ- rpsL nupG/bMON14272/pMON7124 Cellfectin ® Transfection Reagent Cellfectin ® Reagent is supplied with the Bac-to-Bac ® Baculovirus Expression System only. Amount supplied: 1 ml Composition: 1 mg/ml transfection reagent in membrane-filtered water Storage conditions: +4°C vi
Accessory Products Introduction The products listed in this section may be used with the Bac-to-Bac ® Baculovirus Expression System. For more information, refer to our Web site (www.invitrogen.com) or call Technical Service (see page 62). Additional Products All of the reagents supplied in the Bac-to-Bac ® Baculovirus Expression System as well as other products suitable for use with the Bac-to-Bac ® System are available separately from Invitrogen. Ordering information for these reagents is provided below. Item Quantity Catalog no. Bac-to-Bac ® Vector Kit 1 kit 10360-014 Bac-to-Bac ® HT Vector Kit 1 kit 10584-027 pFastBac Dual Vector Kit 1 kit 10712-024 Gateway ® pDEST 8 Vector 6 µg 11804-010 Gateway ® pDEST 10 Vector 6 µg 11806-015 Gateway ® pDEST 20 Vector 6 µg 11807-013 MAX Efficiency ® DH10Bac Competent E. coli 5 x 100 µl 10361-012 One Shot ® TOP10 Chemically Competent E. coli 20 x 50 µl C4040-03 One Shot ® MAX Efficiency ® DH10B -T1 R Chemically Competent E. coli 20 x 50 µl 12331-013 Cellfectin ® Reagent 1 ml 10362-010 Ampicillin 20 ml (10 mg/ml) 11593-019 Kanamycin Sulfate 100 ml (10 mg/ml) 15160-054 Gentamicin 10 ml (50 mg/ml) 15750-060 Tetracycline 5 g Q100-19 Bluo-gal 1 g 15519-028 Isopropylthio-β-galactoside (IPTG) 1 g 15529-019 TEV Protease, Recombinant 1000 units 10127-017 Platinum ® Taq DNA Polymerase 100 reactions 10966-018 Platinum ® Taq DNA Polymerase High Fidelity 100 reactions 11304-011 500 reactions 11304-029 PCR SuperMix High Fidelity 100 reactions 10790-020 M13 Forward (-40) Primer 2 µg N540-02 M13 Reverse Primer 2 µg N530-02 4% Agarose Gel 40 ml 18300-012 CAT Antiserum 50 µl R902-25 continued on next page vii
Page 1: Bac-to-Bac ® Baculovirus Expressio
Page 5: Kit Contents and Storage Types of P
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 and 74:
Purchaser Notification, continued L
Page 75 and 76:
Product Qualification, continued pF
Page 77 and 78:
References Anderson, D., Harris, R.
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Invitrogen Bac to Bac Expression System

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