User Manual
Contents
1. Introduction The BaculoPORTER Transfection Reagent is a lipid formulation of a novel proprietary cationic lipid and dioleoyl phosphatidylethanolamine DOPE in a ratio targeted and optimized for the delivery of DNA into insect cells especially Sf9 and Sf21 When mixed with DNA the BaculoPORTER Transfection Reagent coats and interacts with the DNA to form a lipoplex of lipid and DNA The formed lipoplex is efficient at delivering the target DNA into insect cells and with minimal toxicity The BaculoPORTER Transfection Reagent allows researchers to maximize the delivery of DNA combinations including plasmids transfer vector and baculovirus DNA and bacmid DNA into insect cells to optimize expression and maximize viral titer production The BaculoPORTER Transfection Reagent offers the following benefits Highly efficient delivery of plasmids bacmids and co transfections of a transfer vector and baculovirus DNA into Sf9 and Sf21 cells Simple and straightforward protocol Compatibility with diverse insect cell culture medias Minimized cytotoxicity When compared to other commercially available transfection reagents the BaculoPORTER Transfection Reagent consistently offers superior transfection efficiencies and viral titer production METHODS AND PROCEDURES 1 Transfection of Bacmid DNA into Insect Cells 1 1 Set up the transfections in 35mm tissue culture dishes Seed each plate with 900 000 cells in 2 0 ml of your2. Use DNase free procedures for DNA isolation and purification and plasticware Check the DNA quantity and quality on an agarose gel and or by using a spectrophotometer Poor health of insect Thaw out a fresh aliquot of cells and passage once or a few cells times before transfecting Avoid using cells that have been in culture or have been passaged for excessive periods of time Suboptimal cell Follow the recommendations for cell densities that are density provided in the Materials and Methods Section Alternatively use cells that are 50 70 or 80 confluent on the day of transfection Improper storage The BaculoPORTER Transfection Reagent is very stable but long exposure to elevated temperatures and or excessive freeze thaw cycles may cause degradation of the reagent Store the BaculoPORTER Transfection Reagent at 4 C Wrong medium Be sure to use the correct serum free insect cell culture media when forming the BaculoPORTER transfection mixes BaculoPORTER DNA The BaculoPORTER transfection mixes should be freshly transfection mixes prepared If mixes have been prepared and stored for longer are not freshly than 45 minutes aggregation may occur and transfection prepared efficiencies may be compromised Aggregation Suboptimal Too much BaculoPORTER or too much DNA could cause BaculoPORTER aggregation adjust the ratios as outlined above DNA ratio used BaculoPORTER The BaculoPORTER Transfection Reagent is stable and should
3. frozen and thawed be stored at 4 C at all times Avoid multiple freeze thaw multiple times cycles that may cause lipid aggregation Cytotoxicity Unhealthy cells Check cells for contamination Thaw a new batch of cells Cells are too confluent or cell density too low Check culture medium pH kind used last time changed Check materials used for proper function culture plates incubator temperatures etc BaculoPORTER concentration too high BaculoPORTER is a potent lipid delivery reagent too much lipid however may cause toxicity and if so reduce the total amount of BaculoPORTER by 20 or 30 increments For additional troubleshooting assistance please contact our Technical Support Department Tel 858 457 1919 Ext 3 OR 888 428 0558 Ext 3 US toll free Fax 858 623 9494 E mail techi genlantis com URL www genlantis com Version MV032206 Genlantis 858 457 1919 or 888 428 0558 US Toll Free
4. preferred insect cell culture media with or without serum Allow the cells to incubate for 24 hours at 27 C 1 2 Prepare the BaculoPORTER transfection mix as follows 1 2 1 Prepare DNA Solution by combining 2 0 ug bacmid DNA and serum free media SFM for a total volume of 100 ul in a 1 5ml micro centrifuge tube 1 2 2 Prepare BaculoPORTER solution by combining 5 0 ul of BaculoPORTER Transfection Reagent with 95 ul SFM 1 2 3 Combine the DNA and BaculoPORTER solutions to form the BaculoPORTER transfection mix incubate at room temperature for 15 minutes to allow lipid DNA complexes to form Mix well by pipetting up and down several times 1 2 4 Add 800 ul SFM to the tube bringing the final volume of the BaculoPORTER transfection mix to 1 0ml 1 3 Aspirate the media in which the insect cells are growing in from Step 1 1 above and wash the cells once with 2 ml SFM 1 4 Aspirate the wash and overlay the cells with the 1 0 ml of BaculoPORTER transfection mix prepared in Section 1 2 3 above 1 5 Incubate the cells for 4 hours in a 27 C incubator 1 6 After incubation aspirate the BaculoPORTER transfection mix and replace with 2 0 ml of your preferred insect cell culture media 1 7 Incubate the transfected cells for 5 days at 27 C Version MV032206 Genlantis 5 858 457 1919 or 888 428 0558 US Toll Free 2 Co transfection of Baculovirus DNA and Transfer Plasmid into Insect Cells 2 1 Set up the transfections in
5. 35mm tissue culture dishes Seed each plate with 900 000 cells in 2 0 ml of your preferred insect cell culture media with or without serum Allow the cells to incubate for 24 hours at 27 C 2 2 Prepare the BaculoPORTER transfection mix as follows 2 2 1 Prepare DNA solution by combining 0 5 ug of the baculovirus DNA and 2 0 ug of your transfer vector and serum free media SFM for a total volume of 100 ul in a 1 5ml micro centrifuge tube 2 2 2 2 23 Prepare BaculoPORTER solution by combining 5 0 ul of the BaculoPORTER Transfection Reagent with 95 ul SFM Combine the DNA and BaculoPORTER Solutions to form the BaculoPORTER transfection mix incubate at room temperature for 15 minutes to allow lipid DNA complexes to form Mix well by pipetting up and down several times Add 800ul SFM to the tube bringing the final volume of the BaculoPORTER transfection 2 2 4 mix to 1 0ml 2 3 Aspirate the media in which the insect cells are growing in from Step 2 1 above and wash the cells once with 2 ml SFM 2 4 Aspirate the wash and overlay the cells with the 1 0 ml of BaculoPORTER transfection mix prepared in Section 2 2 3 above 2 5 Incubate the cells for 4 hours in a 27 C incubator 2 6 After incubation aspirate the BaculoPORTER transfection mix and replace with 2 0 ml of your preferred insect cell culture media 2 7 Incubate the transfected cells for 5 days at 27 C 3 Transfection of Baculovirus Expres
6. BaculoPORTER Transfection Reagent Instruction Manual Catalog Numbers T701007 T701035 p Genlantis Genlantis A Division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Phone 888 428 0558 US Toll Free e 858 457 1919 Fax 858 623 9494 e 858 558 3617 E mail orders genlantis com Web Site http www genlantis com TABLE OF CONTENTS Page Purchaser Notification ccccccccseccscceceeccuecuseueeeveueeueeuueaueaueaueaueuueeueaueaueeesueaueausaueaueeuenanes 3 OVERVIEW Kit CONTENTS iss ire diac sesocaott dus eer erase c3 eme L yag A iP qr re seven sario 2od acp AEA ME ENA SUR idR TA 4 Shipping and Storage viii reir eene ee teet ra des rh ain dia eee he ia eed ea eda eed 4 R l ted Products sos ana DOTT AE a a e E ESPASE ASAN 4 Product Support ence A EE EAA a en rv ev su YE Las o E 4 Introducti S METTRE TCTTITOT OT LTLO a a e N Aaaa aa E AT NOSES 5 METHODS AND PROCEDURES Transfection of Bacmid DNA into Insect Cells eee mmn 5 Co transfection of Baculovirus DNA and Transfer Plasmid into Insect Cells 6 Transfection of Baculovirus Expression Plasmid into Insect Cells Grown in 96 well Plates 6 APPENDIX Troubleshooting Guide 5 2 nnne tects etre eR HR e Ea E LOS ERR ER ER 8 Version MV032206 Genlantis 858 457 1919 or 888 428 0558 US Toll Free Purchaser Notification Limited License The purchase price paid for the BaculoP
7. For maximum stability store all reagents at 4 C upon receipt If stored properly all components are stable for 6 months Related Products For efficient transfection of DNA into a wide range of difficult to transfect cell types Product Name Cat No Quantity GenePORTER 2 Transfection Reagent 1202007 75 reactions 0 75 ml GenePORTER 2 Transfection Reagent 1202015 150 reactions 1 5 ml GenePORTER 2 Transfection Reagent 1202075 750 reactions 5 x 1 5 ml For efficient transfection of DNA into neuronal cells Product Name Cat No Quantity NeuroPORTER Transfection Reagent 7400150 1 5 ml NeuroPORTER Transfection Reagent 1400750 5x 1 5 ml For direct delivery of proteins into living cells Product Name Cat No Quantity BioPORTER Protein Delivery Reagent QuikEase Kit BP502424 24 single use vials BioPORTER Protein Delivery Reagent QuikEase Kit BP509696 96 single use vials Product Support To place an order To contact technical support Tel 858 457 1919 Ext 2 OR Tel 858 457 1919 Ext 3 OR 888 428 0558 Ext 2 US toll free 888 428 0558 Ext 3 US toll free Fax 858 623 9494 Fax 858 623 9494 Web http www genlantis com E mail techiGgenlantis com For a complete list of international distributors please visit our website at www genlantis com Version MV032206 Genlantis 4 858 457 1919 or 888 428 0558 US Toll Free
8. ORTER Transfection Reagent kit by end users grants them a non transferable non exclusive license to use the kit and or its separate and included components as listed in the Kit Contents section This kit is intended for internal research only by the purchaser Such use is limited to the transfection of molecules into insect cells as described in the product manual Furthermore research only use means that this kit and all of its contents are excluded without limitation from resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis a division of Gene Therapy Systems Inc GTS Separate licenses are available from GTS for the express purpose of non research use or applications of the BaculoPORTER Transfection Reagent To inquire about such licenses or to obtain permission to transfer or use the enclosed material contact the Director of Licensing at GTS Purchasers may terminate this License at any time by returning all BaculoPORTER Transfection Reagent material and documentation to GTS or by destroying all BaculoPORTER Transfection Reagent kit components Purchasers are advised to contact GTS with the notification that a BaculoPORTER Transfection Reagent kit is being returned in order to obtain a refund and or to expressly terminate a research only license granted through the purchase of the kit s This document covers in full the terms of the BaculoPORTER Tra
9. nsfection Reagent research only license and does not grant any other express or implied license The laws of the State of California shall govern the interpretation and enforcement of the terms of this License Product Use Limitations The BaculoPORTER Transfection Reagent and all of its components are developed designed intended and sold for research use only They are not to be used for human diagnostic or included used in any drug intended for human use All care and attention should be exercised in the handling of the kit components by following appropriate research laboratory practices For more information or for any comments on the terms and conditions of this License please contact Director of Licensing Genlantis a division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Telephone 858 457 1919 Fax 858 623 9494 Email licensing genlantis com Version MV032206 Genlantis 3 858 457 1919 or 888 428 0558 US Toll Free OVERVIEW Kit Contents Catalogue Number of Description Size or Aliquot Number Tubes 1701007 1 tube Hydrated BaculoPORTER lipid 0 75 ml 1701035 5 tubes Hydrated BaculoPORTER lipid 5 x 0 75 ml Each BaculoPORTER Transfection Reagent tube contains sufficient material for 150 transfections based on transfecting 2 ug of DNA in a 35 mm tissue culture dish Shipping and Storage The BaculoPORTER Transfection Reagent is shipped at room temperature
10. or 15 minutes to allow the lipid DNA complexes to form Mix well by pipetting up and down several times 3 2 4 If needed add SFM to the BaculoPORTER transfection mix to bring the final transfection volume up to the recommended value in TABLE I above 3 3 Carefully aspirate the media in which the insect cells are growing in from Step 3 1 above and immediately wash the cells with PBS Aspirate the PBS wash gently 3 4 After washing the cells gently add the BaculoPORTER transfection mix prepared in Section 3 2 3 above 3 5 Incubate the cells for 4 hours in a 27 C incubator 3 6 After incubation aspirate the BaculoPORTER transfection mix and replace with an appropriate volume of your preferred insect cell culture media 3 7 Incubate the transfected cells in a 27 C incubator Heterologous gene expression can normally be detected after 24 hours Version MV032206 Genlantis 7 858 457 1919 or 888 428 0558 US Toll Free APPENDIX Troubleshooting Guide Problem Possible Causes Recommended Solutions Low transfection Suboptimal Optimize the BaculoPORTER DNA ratio by trying any or all of efficiency BaculoPORTER DNA the following ratios 4 1 2 1 1 1 1 2 BaculoPORTER to DNA ratio w v Suboptimal DNA After establishing the optimal BaculoPORTER DNA ratio vary concentration the quantity of DNA by decreasing or increasing few fold 2x and 4x for example to optimize Poor DNA quality
11. sion Plasmid into Insect Cells 3 1 Seed a tissue culture plate with insect cells at the appropriate densities referred to in Table 1 below using your preferred insect cell culture media Allow the cells to incubate for 24 hours at 27 C TABLE 1 Seeding densities and transfection volumes for insect cells grown in different formats Tissue Cell Seeding DNA Amount to BaculoPORTER Final Culture Density per Well Use per Well or Amount to Use Transfection Dish or Plate Plate ug per Well or Volume per Well Plate ul or Plate 96 well 15 000 1 0 1 0 100 ul 24 well 60 000 4 0 4 0 250 ul 6 well OR 240 000 16 0 16 0 1ml 35 mm 60 mm 400 000 25 0 25 0 2 5 ml 100 mm 700 000 45 0 45 0 5ml Version MV032206 Genlantis 6 858 457 1919 or 888 428 0558 US Toll Free 3 2 Depending on the number of cells to be transfected prepare the BaculoPORTER transfection mix as follows 3 2 1 Prepare the DNA Solution by combining the appropriate amount of the baculovirus expression plasmid with serum free media SFM up to half of the recommended transfection volume per well or plate 3 2 2 Prepare the BaculoPORTER Solution by combining the appropriate amount of BaculoPORTER Transfection Reagent with serum free media SFM up to half of the recommended transfection volume per well or plate 3 2 3 Combine the DNA and BaculoPORTER solutions to form the BaculoPORTER transfection mix incubate at room temperature f
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