Bac-to-Bac TOPO Expression System - Thermo Fisher

1 User ManualCorporate HeadquartersInvitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008T: 1 760 603 7200F: 1 760 602 6500E: country-specific contact information visit our web site at Bac-to-Bac TOPO Expression System An efficient cloning and site-specific transposition System to generate recombinant baculovirus for high-level protein Expression Catalog nos. A11101, A11100 Version A 15 December 2008 A10606 ii iii Table of Contents Kit Contents and Accessory Products ..viii Overview ..1 The Bac-to-Bac TOPO Expression System ..3 Experimental Methods .. 7 Culturing Insect Cells ..7 General Generating the Recombinant pFastBac Vector ..9 Generating the Recombinant Bacmid ..10 Transforming DH10 Bac E. Isolating Recombinant Bacmid DNA.

2 14 Analyzing Recombinant Bacmid DNA by PCR ..16 Producing Recombinant Baculovirus ..19 Transfecting Insect Isolating P1 Viral Amplifying Your Baculoviral Stock ..24 Performing a Viral Plaque Assay ..26 Expressing Your Recombinant Analyzing Recombinant Protein ..35 Recipes ..42 Bacmid DNA Isolation Using PureLink HiPure Maxiprep Kit ..44 Technical Purchaser Notification ..48 iv vKit Contents and Storage Types of Products This manual is supplied with the products listed below. For a list of the reagents supplied with each catalog number, see below and the next page. Product quantity Cat. no. Bac-to-Bac C-His TOPO Expression System 1 kit A11100 Bac-to-Bac N-His TOPO Expression System 1 kit A11101 Kit Components Each Bac-to-Bac TOPO Expression System contains the components listed below.

3 See the next page for a detailed description of other reagents supplied with each System . Component Cat. no. A11100 Cat. no. A11101 Bac-to-Bac C-His TOPO Cloning Kit Bac-to-Bac N-His TOPO Cloning Kit One Shot Mach1-T1R Chemically Competent E. coli MAX Efficiency DH10 Bac Competent E. coli Cellfectin II Reagent Bac-to-Bac TOPO Cloning Kit Manual Bac-to-Bac TOPO Expression System Manual Shipping/Storage The Bac-to-Bac C-His or N-His TOPO Expression System is shipped in four boxes as described below. Upon receipt, store each box as detailed below. All reagents are guaranteed for six months if stored properly. Box Item Shipping Storage 1 Bac-to-Bac C-His TOPO or Bac-to-Bac N-His TOPO Cloning Kit Dry ice 20 C 2 One Shot Mach1-T1R Chemically Competent E.

4 Coli Dry ice 80 C 3 MAX Efficiency DH10 Bac Competent E. coli Dry ice 80 C 4 Cellfectin II Reagent Gel ice 4 C Continued on next page vi Kit Contents and Storage, continued Bac-to-Bac TOPO Cloning Kits The cloning reagents for the Bac-to-Bac N-His and C-His TOPO Cloning Kits (Box 1) are listed below. Store the contents of Box 1 at 20 C. Item Concentration Amount pFastBac /NT-TOPO vector (Cat. no. A11101) or pFastBac /CT-TOPO vector (Cat. no. A11100) 20 l at 10 ng/ l in 50% glycerol 50 mM Tris-HCl, pH (at 25 C) 1 mM EDTA 2 mM DTT Triton X-100 100 g/ml BSA 30 M bromophenol blue 20 l 10X PCR Buffer 100 mM Tris-HCl, pH (at 42 C) 500 mM KCl 25 mM MgCl2 gelatin 100 l dNTP Mix mM each dATP, dCTP, dGTP, and dTTP.

5 Neutralized at pH in water 10 l Salt Solution M NaCl M MgCl2 50 l Sterile Water 1 ml Control PCR template 50 ng/ l in TE buffer*, pH 10 l Control PCR primers 100 ng/ l each in TE buffer, pH 10 l Polyhedrin forward sequencing primer 100 ng/ l in TE buffer, pH 20 l SV40 polyA reverse sequencing primer 100 ng/ l in TE buffer, pH 20 l pFastBac Gus ng/ l in TE buffer, pH 20 l *TE buffer, pH : 10 mM Tris HCl, 1 mM EDTA, pH Continued on next page viiKit Contents and Storage, continued One Shot Mach1 T1R Competent E. coli The following reagents are included in the One Shot Mach1 T1R Chemically Competent E. coli kit (Box 2). Transformation efficiency of One Shot Mach1 T1R E. coli cells is 1 109 cfu/ g DNA. Store cells at 80 C. Reagent Composition Amount One Shot Mach1 T1R Chemically Competent E.

6 Coli 21 50 l Medium (may be stored at room temperature or 4 C) 2% tryptone yeast extract 10 mM NaCl mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose 6 ml pUC19 Control DNA 10 pg/ l in 5 mM Tris-HCl, mM EDTA, pH 50 l Genotype of Mach1 T1R F 80(lacZ) M15 lacX74 hsdR(rK mK+) recA1398 endA1 tonA MAX Efficiency DH10 Bac Competent E. coli MAX Efficiency DH10 Bac Competent E. coli (Box 3) have a transformation efficiency of 1 x 108 cfu/ g DNA. Store at 80 C. Item Composition Amount MAX Efficiency DH10 Bac Competent E. coli 4 kits (4 5 reactions) pUC19 Control DNA 10 pg/ l in 5 mM Tris-HCl, mM EDTA, pH 100 l Genotype of DH10 Bac F mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 endA1 araD139 (ara, leu)7697 galU galK rpsL nupG/bMON14272/pMON7124 Cellfectin II Transfection Reagent Cellfectin II Reagent is a proprietary cationic lipid formulation that offers the highest transfection efficiencies and protein Expression levels on the widest variety of adherent and suspension insect cell lines.

7 Amount supplied: 1 ml Composition: 1 mg/ml transfection reagent in membrane-filtered water Storage conditions: 4 C (do not freeze) viii Accessory Products Introduction The products listed in this section may be used with the Bac-to-Bac C-His and N-His TOPO Expression Systems. For more information, refer to our website at or contact Technical Support (see page 47). Additional Products All of the reagents supplied in the Bac-to-Bac TOPO Expression System as well as other products suitable for use with the Bac-to-Bac TOPO Expression System are available separately from Invitrogen. Ordering information for these reagents is provided below. Item quantity Cat. no.

8 Bac-to-Bac C-His TOPO Cloning Kit 1 kit A10636 Bac-to-Bac N-His TOPO Cloning Kit 1 kit A10637 MAX Efficiency DH10 Bac Competent E. coli 5 100 l 10361-012 One Shot Mach1 -T1R Chemically Competent E. coli 21 50 l C8620-03 Cellfectin II Reagent 1 ml 10362-100 Platinum Pfx DNA Polymerase 100 units 11708-013 AccuPrime Pfx DNA Polymerase 200 reactions 12344-024 Pfx50 DNA Polymerase 100 reactions 12355-012 Platinum Taq DNA Polymerase High Fidelity 100 reactions 11304-011 PureLink PCR Purification Kit 50 preps K3100-01 PureLink Quick Gel Extraction System 1 kit K2100-12 PureLink HiPure Plasmid Miniprep Kit 25 preps 100 preps K2100-02 K2100-03 PureLink HiPure Plasmid Midiprep Kit 25 preps 50 preps K2100-04 K2100-05 PureLink HiPure Plasmid Maxiprep Kit 10 preps 25 preps K2100-06 K2100-07 Ampicillin Sodium Salt, irradiated 200 mg 11593-027 Kanamycin Sulfate 5 g 25 g 11815-024 11815-032 Kanamycin Sulfate (100X), liquid 100 ml 15160-054 Gentamicin Reagent Solution, liquid (50 mg/ml)

9 10 ml 10 10 ml 15750-060 15750-078 Bluo-gal 1 g 15519-028 Isopropylthio- -galactoside (IPTG) 1 g 15529-019 Medium 10 10 ml 15544-034 (Miller s LB Broth Base) Luria Broth Base, powder 500 g 12795-027 Continued on next page ixAccessory Products, continued Additional Products, continued All of the reagents supplied in the Bac-to-Bac TOPO Expression System as well as other products suitable for use with the Bac-to-Bac TOPO Expression System are available separately from Invitrogen.

10 Ordering information for these reagents is provided below. Item quantity Cat. no. Water, distilled (cell-culture grade) 500 ml 15230-162 BaculoTiter Assay Kit 30 titers K1270 4% Agarose gel (optimal for insect cell growth) 40 ml 18300-012 Fetal Bovine Serum (FBS), Qualified, Heat Activated 100 ml 16140-063 Insect Cell Culture Products A variety of insect cell lines and GIBCO cell culture products are available from Invitrogen to facilitate baculovirus-mediated Expression of your recombinant protein in insect cells. For more information about the insect cell lines and GIBCO cell culture products, refer to our website at or contact Technical Support (see page 47).