Transfection Reagent
Contents
1. 500 ul 20 ug ml oO w lm Add the appropriate volume of complexes solution directly to your cells as illustrated below DNA NP Complex DNA Vol Setu Well Transfer p ug Well s 2000000 w20 00000 2 0 1000 uC O O C O 3 0 1500 E O O O C ll Optimization conditions for neuronal cell line transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We recommend using the DNA Diluent for neuronal cell lines such as NT2 Setup DNA dilutions NeuroPORTER Total Final DNA in SFM dilutions in SFM Volume Concentration 1 12 5 ul in 112 5 yl 2 25 ul in 100 ul 3 10 ugin 50 ul in 75yl 4 125 75ulin50oui 0H 40 ugm 5 100 ul in 25 ul 6 125 uI NP Only SFM Serum free medium Add the appropriate volume of complexes solution directly to your cells as illustrated below VKM110906 Page 5 of 6 DNA NP Complex DNA Vol Setup Mell Transfer ug Well 1 05 125 yl mC O 1 0 250p C O 20 50 0pl O u me 00000 lll Optimization conditions for differentiated post mitotic neurons and glial cell line transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We do not recommend using the DNA Diluent for differentiated post mitotic neurons and glial cells Setup DNA dilutions NeuroPORTER Total Final2. 2 5 2 6 NOTE Although NeuroPORTER has been optimized for specific cell culture conditions optimization may be needed to achieve maximum transfection efficiency The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization of the ratio of NeuroPORTER reagent to DNA start by using 2 5 to 15 ul of reagent for each 1 ug of DNA Use a fixed amount of DNA or vary the amount as suggested in the Appendix to optimize this ratio Dilute the DNA with the serum free medium do not use the DNA Diluent for primary neurons Refer to Table 4 for the appropriate volume of serum free medium NOTE To obtain maximum efficiency in particular cells some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization of the DNA quantity used maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over a suggested range see Table 5 See the Appendix for examples Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form NOTE Do not incubate the DNA solution with the NeuroPORTER Transfection Reagent for longer than 30 minutes Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 5 for suggested medium volumes Table 5
3. Medium Volumes and DNA Amount for Various Culture Dishes DNA Plating Medium ug Volume ml 0 1 0 5 0 2 0 5 3 0 0 5 1 0 4 0 1 0 2 0 6 0 1 5 6 0 8 0 2 5 8 0 12 0 5 0 Add fresh growth media as needed 24 hours post transfection Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours following transfection NOTE For some cell types the old media can be replaced with fresh media at this step 3 Transfection of Neuronal Cell Lines 3 1 3 2 3 3 Hydrate NeuroPORTER lipid film at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 6 for the appropriate volume of serum free medium Table 6 Volumes of Transfection Reagents DNA DNA Neuro Serum Free Medium ug Diluent PORTER for NeuroPORTER ul ul ul 0 5 6 25 1 25 5 0 1 0 12 5 2 5 10 0 2 0 25 0 5 0 20 0 4 0 50 0 10 0 40 0 Dilute the DNA with the DNA Diluent and incubate 1 to 5 minutes at room temperature Refer to Table 6 for the appropriate volume of DNA Diluent Do not incubate DNA with the DNA Diluent for longer than 5 minutes Avoid vortexing the DNA diluent NOTE Although NeuroPORTER consistently delivers high transfection efficiencies in o
4. Transfection Reagent is a novel cationic lipid specially formulated for optimal transfection in neuronal cells including primary neurons differentiated post mitotic neurons neuronal cell lines and glial cells NeuroPORTER Transfection Reagent is much easier to use than the traditional viral delivery method for transfecting DNA into neuronal cells NeuroOPORTER Transfection Reagent is compatible with serum eliminates the need to change media following transfection An included DNA Diluent is designed to facilitate DNA lipid complex lipoplex formation and enhance the transformation efficiency in certain neuronal cells such as NT2 not recommended for primary and differentiated neurons Compared to other commercially available transfection reagents NeuroPORTER provides superior transfection efficiency and minimized cytotoxicity Cell type specific protocols are developed for NeuroPORTER Transfection Reagents to ensure optimal transfection results Methods and Procedures 1 Transfection of Primary Rat Hippocampal Neurons 1 1 1 2 Seed primary rat hippocampal cells in poly D lysine coated plates Becton Dickinson Labware in the numbers listed in Table 1 below using the following Plating Medium Neurobasal medium Invitrogen Cat No 21103 049 supplemented with B27 0 5 mM L glutamine and 25 uM glutamate Incubate the cells at 37 C in 5 CO2 for 72 hours Table 1 Suggested Cell Plating Numbers Cell Number Pl
5. DNA ul ul Neuro PORTER ul 0 5 15 0 5 0 10 0 1 0 25 0 10 0 15 0 2 0 50 0 20 0 30 0 40 75 0 40 0 35 0 6 0 100 0 60 0 40 0 8 0 150 0 80 0 70 0 Dilute the DNA with the serum free medium Refer to Table 9 for the appropriate volume of serum free medium NOTE Although NeuroPORTER consistently delivers high transfection efficiencies in order to obtain maximum efficiency in particular cell types some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization first maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over the suggested range If necessary optimize the ratio of NeuroPORTER reagent to DNA by using 5 to 20 ul of reagent for each 1 ug of DNA Use a low DNA quantity to optimize this ratio Following this process cell numbers can also be optimized See the Appendix for examples Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate the DNA solution with the NeuroPORTER Transfection Reagent for longer than 30 minutes Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 10 for suggested cell number according to culture dishes size and cell types Refer to Table 11 for appropriate medium volumes Cells plated
6. A Serum Free Total ug Medium Transfection Volume ml Volume ml 0 1 0 5 0 1 0 125 1 0 3 0 0 45 0 5 2 0 4 0 0 925 1 0 4 0 6 0 1 375 1 5 1 6 Remove the Plating Medium from the cells and add the volume of serum free medium indicated in Table 3 to each well 1 7 Apply the DNA NeuroPORTER complexes from step 1 5 to each well The total transfection volume at this step is indicated in Table 3 1 8 Gently mix the DNA NeuroPORTER serum free medium by swirling and place the cells in a 37 C incubator with 5 C02 1 9 After two hours of incubation add one additional volume of fresh Culture Medium containing 2X concentration of B27 onto the cells 1 10 Perform assay for gene expression after 24 48 hours 2 Transfection of Other Primary Neurons 2 1 Hydrate the NeuroPORTER lipid vial at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use 2 2 Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 4 for the appropriate volume of serum free medium Table 4 Volumes of Transfection Reagents DNA Serum Free Neuro Serum Free ug Medium for PORTER Medium for Neuro DNA ul ul PORTER ul 0 5 12 5 2 5 10 0 1 0 20 0 5 0 15 0 2 0 40 0 10 0 30 0 4 0 55 0 20 0 35 0 6 0 70 0 30 0 40 0 8 0 110 0 40 0 70 0 VKM110906 Page 2 of 6 2 3 24
7. DNA in SFM dilutions in SFM Volume Concentration 50 ul in 200 ul 75 ul in 175 pl 10 ug in 100 yilin 150 pl 250 ul 125 ulin 125 ul 00M 150 ul in 100 yl 200 pl in 50 pl 20 ug ml Add the appropriate volume of complexes solution directly to your cells as illustrated below DNA NP Complex DNA Vol Setu Well Transfer p ug Well 1 5 sw gt O 10 sw BO O 20 wB C 30 wu O C C OC O Quality Control To assure the performance of each lot of the NeuroPORTER reagent we pre qualify the chemical synthesis of NeuroPORTER lipid by mass spectrometry and thin layer chromatography The final product is further tested by in vitro B galactosidase transfection assay in NT2 neuronal precursor cell Each lot shall have an acceptance specification of gt 70 of the activity of the Reference lot QC 3 OOO VKM110906 Page 6 of 6
8. NeuroPORTER GenePORTER 3000 Transfection Reagent Genlantis A division of Gene Therapy Systems Inc 7203007 0 75 ml 107 reactions 7203015 1 5 ml 214 reactions 7203115 1 5 ml 2 140 reactions GenePORTER Gold Transfection Reagent 7204015 1 5 ml 400 reactions 7204030 2 x 1 5 ml 800 reactions 7204115 15 ml 4 000 reactions Shipped at room temperature Store kit at 4 C T400101S NeuroPORTER Transfection Reagent 0 2 ml Trial Size DNA Diluent 1 0 ml T400150 NeuroPORTER Transfection Reagent 1 vial 75 300 Dried Lipid Film rxns Hydration Buffer 1 5 ml DNA Diluent 7 5 ml T400750 NeuroPORTER Transfection Reagent 5 vials 375 1 500 Dried Lipid Film rxns Hydration Buffer 5x 1 5 ml DNA Diluent 5x7 5ml GenePORTER 2 Transfection Reagent 7202007 0 75 ml 75 reactions 7202015 1 5 ml 150 reactions 7202075 5 x 1 5 ml 750 reactions GeneSilencer siRNA Transfection Reagent 7500750 0 75 ml 200 reactions 7505750 5 x 0 75 ml 1 000 reactions BioPORTER Protein Delivery Reagent BP502424 24 single use tubes BP509696 96 single use tubes NeuroFect Transfection Reagent T800075 0 75 ml 75 300 reactions 7800750 5 x 0 75 ml 375 1500 rxns MycoScope PCR Mycoplasma Detection Kit MY01050 50 reactions MY01100 100 reactions Introduction NeuroPORTER
9. ating Medium per well Volume 15 000 0 125 ml 100 000 0 5 ml 200 000 1 0 ml 500 000 2 0 ml After 72 hours of incubation remove 1 2 volume of the Plating Medium and replace with the following Culture Medium Neurobasal medium supplemented B27 and 0 5 mM L glutamine no 25 uM glutamate Continue incubation for an additional 24 hours VKM110906 1 3 Page 1 of 6 Hydrate the NeuroPORTER lipid vial at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use Dilute the DNA and hydrated NeuroPORTER reagent with serum free medium do not use the DNA Diluent for primary neurons Refer to Tables 2 and 3 for recommended DNA NeuroPORTER and serum free medium volumes for different tissue culture plates Table 2 Volumes of Transfection Reagents s F Serum Free DNA erum free Neuro Medium For Medium for ug DNA ul PORTER ul Neuro PORTER il 0 1 0 5 12 5 2 5 10 0 1 0 3 0 25 0 5 0 20 0 2 0 4 0 37 5 7 5 30 0 4 0 6 0 62 5 12 5 50 0 1 5 Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Mix by pipetting up and down several times Incubate at room temperature for 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate for longer than 30 minutes Table 3 Medium Volumes and DNA Amount for Various Culture Dishes DN
10. dium containing the appropriate selection antibiotic It is important to wait at least 48 hours before exposing the transfected cells to the selection media For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition 3 4 Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate the DNA solution with the 4 1 Hydrate NeuroPORTER lipid film at room temperature with NeuroPORTER Transfection Reagent for longer than 30 1 5 ml of the hydration buffer Vortex for 30 60 seconds at minutes top speed Store the hydrated reagent at 4 C and vortex briefly before use 4 Transfection of Differentiated Post Mitotic Neurons and Glial Cell Lines 3 5 Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 7 for suggested cell numbers for specific tissue culture dishes Refer to Table 8 for appropriate medium volumes NOTE Cells plated the day before transfection should be 50 to 70 confluent on the day of transfection 4 2 Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 9 for the appropriate volume of serum free medium VKM110906 Page 3 of 6 4 3 4 4 4 5 VKM110906 Table 9 Volumes of Transfection Reagents DNA Serum Free Neuro PAA Medium for PORTER ug
11. rder to obtain maximum efficiency in particular cell types some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization first maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over the suggested range If necessary optimize the ratio of NeuroPORTER reagent to DNA by using 1 25 to 12 5 0l of reagent for each 1 Ig of DNA Use a low DNA quantity to optimize this ratio Following this process cell number can also be optimized See the Appendix for examples Table 7 Suggested Cell Culture Conditions for Transfection of Neuronal Cell Lines Number of Cells Well 25 0 30 0 x 10 125 0 150 0 x 10 250 0 300 0 x 10 500 0 600 0 x 10 1 0 1 5 x 108 2 5 3 0 x 10 Table 8 Medium Volumes and DNA Amount for Various Culture Dishes DNA Medium Volume ug ml 0 1 0 5 0 2 0 5 3 0 5 1 0 4 0 1 0 2 0 6 0 1 5 6 0 8 0 2 5 8 0 12 0 5 0 3 6 Add fresh growth media as needed 24 hours post transfection Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours following transfection NOTES For some cell types the old media can be replaced with fresh media at this step The same protocol can be used to produce stably transfected cells 48 to 72 hours post transfection put the cells in fresh me
12. the day before transfection should be 50 to 70 confluent on the day of transfection Table 10 Suggested Cell Culture Conditions for Transfection of Differentiated Neurons and Glial Cells Cells Well Cells Well Diff Neurons Glial Cells 35 x 108 50 x 108 150 x 103 200 x 108 300 x 108 400 x 108 600 x 10 800 x 10 1 5 x 108 2 x 108 100mm 3x 108 4x 108 Page 4 of 6 Table 11 Medium Volumes and DNA Amount for Various Culture Dishes DNA Medium Volume Ug ml 0 1 0 5 0 2 0 5 3 0 0 5 1 0 4 0 1 0 2 0 6 0 1 5 6 0 8 0 2 5 8 0 12 0 5 0 4 6 24 hours post transfection add fresh growth media as needed Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours following transfection NOTE For some cell types the old media can be replaced with fresh media at this step Also the same protocol can be used to produce stably transfected cells 48 to 72 hours post transfection put the cells in fresh medium containing the appropriate selection antibiotic It is important to wait at least 48 hours before exposing the transfected cells to the selection media For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition LIMITED LICENSE The purchase price paid for the NeuroPORTER Transfection Reagent Kit hereto NeuroPORTER grants end users a non
13. transferable non exclusive license to use the kit and or its components for internal research use only as described in this manual in particular research use only excludes and without limitation 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 for non research use or applications NeuroPORTER and or its components are not to be used for human diagnostic or included used in any drug intended for human use Care and attention should be exercised in handling the kit components by following appropriate research laboratory practices and kit instructions Purchasers may refuse this license by returning the enclosed materials unused By keeping or using this kit you agree to be bound by the terms of this license as governed and enforced by the laws of the State of California APPENDIX Transfection Optimization Examples Optimization conditions for primary neuron transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We do not recommend using the DNA Diluent for primary neurons Setup DNA dilutions NeuroPORTER Total in SFM dilutions in SFM Volume 25 ul in 225 ul 50 ul in 200 ul 10 ugin 75 ul in 175pl 250 ul 100 yl in 150 ul 125 ul in 125 ul 6 150 ul in 100 ul SFM Serum free medium Final DNA Concentration
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