Invitrogen Bac to Bac Expression System

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Isolating Recombinant Bacmid DNA, continued DNA Isolation Procedure, continued 12. Remove as much of the supernatant as possible, taking care not to disturb the pellet. Air dry the pellet for 5 to 10 minutes at room temperature. Do not overdry the pellet. Important Note: Do not use a SpeedVac to dry the DNA pellet as this may shear the DNA. 13. Dissolve the DNA pellet in 40 µl of 1X TE Buffer, pH 8.0. To avoid shearing, do not mechanically resuspend the DNA. Allow the solution to sit in the tube with occasional gentle tapping of the bottom of the tube. 14. Store the DNA at +4°C. Note: We do not recommend storing the purified bacmid DNA at -20°C as repeated freezing and thawing may shear the DNA. 15. Proceed to analyze the recombinant bacmid DNA (see Analyzing Recombinant Bacmid DNA by PCR, page 23) or to transfect the DNA into insect cells (see Transfecting Insect Cells, page 26). 52
Map and Features of pFastBac 1 pFastBac 1 Map The map below shows the elements of pFastBac 1. The complete sequence of pFastBac 1 is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 62). BamH I Rsr II BssH II EcoR I Stu I Sal I Sst I Spe I Not I Nsp V Xba I Pst I Xho I Sph I Kpn I Hind III Gentamicin P PH SV40 pA Tn7L Tn7R pFastBac TM 1 4775 bp ori f1 Comments for pFastBac TM 1 4775 nucleotides pUC ori Ampicillin f1 origin: bases 2-457 Ampicillin resistance gene: bases 589-1449 pUC origin: bases 1594-2267 Tn7R: bases 2511-2735 Gentamicin resistance gene: bases 2802-3335 (complementary strand) Polyhedrin promoter (P PH ): bases 3904-4032 Multiple cloning site: bases 4037-4142 SV40 polyadenylation signal: bases 4160-4400 Tn7L: bases 4429-4594 continued on next page 53
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: Isolating Recombinant Bacmid DNA In
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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