Transfection Reagent
Contents
1. Cell Lines DNA Diluent DNA Diluent B Serum HeLa S3 j pau O 2 Use 25 ul of the DNA Diluent or DNA Diluent B per each 1 ug of DNA HeLa k kk O Avoid vortexing either of the DNA diluent solutions COS 1 x C PROTOCOL FOR USING THE DNA DILUENT COS 7 o 3 For most cell types use 5 ul GenePORTER 2 per 1 ug of DNA Hep G2 o 4 Your DNA can be suspended in TE buffer or purified water A DNA NIH 3T3 concentration of at least 0 1 mg ml works well for most reaction sizes MDCK kk O K 562 kk O i Transfection of Adherent Cells CV 1 Ld 5 Dilute the hydrated GenePORTER 2 reagent with serum free medium B15 F0 as shown in Table 1 below 293 o 6 Dilute the DNA with DNA Diluent as shown in Table 1 below mix well BHK 21 x e by pipetting and incubate at room temperature for 1 5 minutes CHO K1 wa wa NOTE Do not vortex DNA Diluent PC 12 ua NR bd Table 1 Amounts of DNA diluent GenePORTER 2 and Medium P19 lal e DNA DNADiluent GenePORTER2 Serum free HUVEC C al al ug w mi Medium u Jurkat e O 0 5 12 5 2 5 10 0 Legend 1 0 25 0 5 0 20 0 Works well O Works well w o serum 2 0 50 0 10 0 40 0 x Works better Works well w and w o serum 40 100 0 20 0 80 0 NR Not recommended Use GenePORTER Reagent 80 200 0 40 0 160 0 4 Best expression is w o serum during 1 hr of transfection VKMO061025 Genlantis Page 1 of 3 Telephone 858 457 1919 888 428 05582. Secreted Alkaline Phosphatase When heat stable secreted alkaline phosphatase SEAP is the reporter gene used for transfections use the following assay heat supernatants from transfected cells at 65 C for 30 min to inactivate endogenous alkaline phosphatase activity The SEAP transgene is stable during this treatment Take aliquots of the culture media 48 hours posttransfection and determine the SEAP activity quantitatively by using a colorimetric assay based on hydrolysis of the chromogenic substrate para nitrophenyl phosphate PNPP Dissolve 1 mg ml of PNPP reagent in a solution of 1 mM MgClzand 100 mM diethanolamine pH 9 8 Add 10 ul of 0 05 Zwittergent in PBS free Ca2 and Mg into each well of a 96 well plate Then add 20 ul of the heated cell culture media to each well For control wells 20 ul of water is used to normalize the volume An alkaline phosphatase standard EIA grade calf intestine alkaline phosphatase Boehringer Mannheim can be used to generate a standard curve from 10 to 10 000 pg per well Add 200 ul of the PNPP substrate to each well to start the enzymatic reaction Allow the reaction to incubate at room temperature until the yellow color develops Using 0 05 Zwittergent in PBS as the diluent virtually reduces the background to zero which increases the detection limit of the assay Read the plates at 405 nm using either kinetic or static mode REFERENCES Felgner JH et al 1994 Enhanced gene delivery and m
3. fecti fS ion Cell For suspension cells the protocol is the same as described for Mo lransrEcton Or SUSPENSION GENS adherent cells with the following exceptions Same as Section ii protocol for transfection of suspension cells when 44 Th F using the DNA Diluent e day before transfection split the cells so they are in good condition on the day of transfection 12 While the GenePORTER 2 DNA complexes are incubating spin E DETECTION OF EXPRESSED REPORTER GENES down the cells resuspend them at 1 x 10 or 2 x 108 cells ml in B Galactosidase medium with or without serum and transfer the appropriate volume The following protocol is provided for your convenience alternatively to the dish as indicate in Table 3 below you could use one of the B galactosidase assay kits offered by 13 Add the GenePORTER 2 DNA complexes directly to the cells and Genlantis Catalog Numbers A10100K A10200K and A10300K mix well by gently pipetting 2 to 3 times NOTE this step is important because some suspension cells have a Briefly aspirate the culture media post transfection Lyse the tendency to clump which reduces transfection efficiency transfected cells from each well of a 96 well plate with 50 ul of the lysis 14 Incubate at 37 C and proceed as described for adherent cells buffer 0 1 Triton X 100 wiv in 250 mM Tris HCl pH 8 0 then NOTE for some hematopoietic cell lines mitogenic agents like PHA Subject the cells to one freeze thaw cycle freeze a
4. U S Toll free e Fax 858 623 9494 www genlantis com Add the diluted DNA to the diluted GenePORTER 2 reagent Incubate Table 3 Number of Cells and Transfection Volumes for Suspension at room temperature for 5 to 10 minutes to form lipid DNA complexes Cells lipoplexes Tissue Culture Number of Cells Transfection NOTE Do not incubate for more than 30 minutes Dish Size ug Volume ml 2 5 8 Add the GenePORTER 2 DNA complexes directly to cells growing in a a i serum containing medium and incubate at 37 C Use Table 2 below 6 well 2 408 1 0 for recommended transfection volumes and DNA amounts We x P NOTES Cells plated the day before transfection should be 50 60 mm 5x10 2 5 70 confluent on transfection day Omitting antibiotics from the 100 mm 1x 107 9 0 media during transfection may increase expression levels this effect is cell type dependent and usually small D PROTOCOL FOR ING THE DNA DILUENT B For some cells such as HeLa S3 MDCK CHO K higher s USING a transfection efficiencies can be achieved when the initial 4 hour 15 For most cell types use 3 5 ul of the GenePORTER 2 reagent per 1 incubation is done in serum free media followed by the addition of g of DNA one volume of medium containing 20 serum Table 2 Transfection Volumes and DNA Amounts Per Dish Size ii Transfection of Adherent Cells Tissue Culture DNA Transfection Volume _ 16 Dilute the hydrated GenePORTER 2 reagent with serum free Dish S
5. echanism studies with a novel series of cationic lipid formulations J Biol Chem 269 2550 2561 Felgner PL et al 1987 Lipofection a highly efficient lipid mediated DNA transfection procedure Proc Nat Acad Sci USA 84 7413 7417 Gussoni E et al 1996 A method to codetect introduced genes and their products in gene therapy protocols Nature Biotechnology 14 1012 1015 Cheng L et al 1996 Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells Nature Biotechnology 14 606 609 1 2 3 4 OPTIONAL PROTOCOL FOR LOW QUANTITY DNA TRANSFECTION The following revised protocol 2 can be used to facilitate pipetting and transfer of DNA lipids complexes to the cells when a low quantity of DNA lt 1 ug is used for transfection 1 Dilute hydrated GenePORTER 2 reagent with serum free medium as in Table 5 below 2 First dilute the DNA diluent in serum free medium and then add the DNA See Table 5 A B and C below for volumes of serum free medium DNA diluent and DNA amounts Table 5 Recommended Amounts of Reagents for Optional Protocol A Dilution of GenePORTER 2 Reagent DNA Serum free ug Medium ul 0 125 0 25 0 5 1 0 GenePORTER 2 Reagent ul B DNA Dilution Serum free Medium ug DNA Diluent 46 8 43 75 37 5 25 0 Transfection Volume ml Tissue Culture Dish 96 well 24 well 3 Incubate 1 to 5 minutes at room temperature 4 Proceed as
6. enePORTER 2 Transfection Reagent 3S Genlantis A division of Gene Therapy Systems Inc Catalog Contents Quantity _Related Products Catalog T202001S GenePORTER 2 Lipid Trial Size 0 1 ml GenePORTER Gold Transfection Reagent 400 reactions T204015 10 reactions DNA Diluent 0 5 ml GenePORTER 3000 Transfection Reagent 107 reactions 1203007 DNA Diluent B 0 5 ml GenePORTER 2 Transfection Reagent 150 reactions 7202015 T202007 GenePORTER 2 Lipid Film dried 1 vial GenePORTER 2 Transfection Reagent 750 reactions T202075 75 reactions Hydration Buffer Transfection Grade 1x 0 8 ml GenePORTER Transfection Reagent 75 reactions 7201007 DNA Diluent 1x4ml GenePORTER Transfection Reagent 150 reactions 7201015 DNA Diluent B 1x4ml GenePORTER Transfection Reagent 750 reactions T201075 7202015 GenePORTER 2 Lipid Film dried 1 vial BoosterExpress Reagents 3 x 1 5 ml T20100B 150 Hydration Buffer Transfection Grade 1x 1 6 ml GeneSilencer siRNA Transfection Reagent 200 reactions 1500750 reactions DNA Diluent 1x8 ml GeneSilencer siRNA Transfection Reagent 1000 reactions 7505750 DNA Diluent B 1x8ml NeuroFect Transfection Reagent 75 300 reactions T800075 7202075 GenePORTER 2 Lipid Film dried 5 vials NeuroFect Transfection Reagent 375 1500 reactions T800750 750 Hydration Buffer Transfection Grade 5x 1 6 ml BioPORTER Protein Delivery Reagent 24 reacti
7. in Steps 6 though 9 under the Transfection of Adherent Cells Section when using the DNA Diluent LIMITED LICENSE The purchase price paid for the GenePORTER 2 Transfection Reagent Kits hereto GenePORTER 2 grants end users a non transferable non exclusive license to use the kits and or their 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 GenePORTER 2 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 Purchasers may refuse this license by returning the enclosed materials unused By keeping or using the enclosed materials you agree to be bound by the terms of this license The laws of the State of California shall govern the interpretation and enforcement of the terms of this License VKM110902 Genlantis Phone 858 457 1919 888 428 0558 U S Toll free e Fax 858 623 9494 Page 3 of 3 www genlantis com
8. ize u9 i ml medium as shown in Table 4 below 96 well 0 1 0 5 0 1 17 Dilute the DNA with DNA Diluent B as shown in Table 4 below mix k well by pipetting and incubate at room temperature for 5 minutes i ees NOTE Do not vortex DNA Diluent B 6 well 2 0 6 0 1 0 60 mm 6 0 8 0 2 5 Table 4 Amounts of DNA diluent GenePORTER 2 and Medium 100 mm 8 0 12 0 5 0 9 Add fresh growth media as needed 24 hours post transfection for some cell types the old media can be replaced with fresh media at this step 10 Depending on cell type and promoter activity reporter gene assay can be performed 24 72 hours post transfection NOTE the same protocol can be used to produce stably transfected cells 48 to 72 hours post transfection put the cells in fresh medium j Pile ds 18 Add the diluted DNA to the diluted GenePORTER 2 Incubate at containing the appropriate selection antibiotic Wait at least 48 hours fae b room temperature for 5 minutes to form lipid DNA complexes efore exposing the transfected cells to the selection medium and lipoplexes o cell types it may be necessary to wait as long as 4 to 5 NOTE Do net incubatefor mote than 30 minutes l i 19 Same as Step 8 above ii Transfection of Suspension Cells 20 Same as Step 9 above GenePORTER 2 reagent works well for most suspension cells such 4 Step 10 ab as K562 and PC 12 but for Jurkat cells we recommend using the SAME AS DIPS ADOND original GenePORTER Reagent W T
9. ons BP502401 reactions DNA Diluent 5x8 ml BioPORTER Protein Delivery Reagent QuikEase kit 24 rxns BP502424 DNA Diluent B 5x8 ml Shipping Shipped at room temperature Storage Store at 4 C stable for 1 year Introduction The GenePORTER 2 transfection reagent is the most effective and widespread gene delivery reagent developed by Genlantis While offering all of the advantages of DHC technology as the original GenePORTER Reagent the GenePORTER 2 Reagent delivers higher transfection efficiencies for more difficult to transfect cell types With a choice of two optimized DNA diluent buffers GenePORTER 2 performs effectively across a broad range of cells offers the highest efficiencies and is effective in the presence of serum eliminating inconvenient media changes METHODS AND PROCEDURES A DNA DILUENT SELECTION B GENEPORTER LIPID HYDRATION Select the DNA diluent suitable for your cell type as listed below if 4 Hydrate each GenePORTER 2 lipid vial at room temperature with 0 75 not listed choose the cell type that closely matches yours or consult ml for T202007 1 5 ml for T202015 and T202075 of the Hydration our citations list at www genlantis com If no data is available for Buffer Vortex for 10 seconds at top speed before use Store hydrated your cell type try the DNA Diluent B protocol first Section C reagent at 4 C vortex briefly before each use Note that Trial Size T202001S is provided in hydrated format
10. t 70 C and thaw at or PMA may be added to the cells 4 hours after transfection to a final room temperature While the cells are being lysed prepare a B concentration of 1 ug ml or 50 ng ml respectively to enhance the galactosidase E coli Sigma standard curve with 0 5 BSA in PBS levels of gene expression w v Once the plate of lysed cells is completely thawed transfer a 50 ul aliquot of each point on the standard curve to control wells of the plate Typically B galactosidase expression ranges from 10 000 to 2 000 000 pg Develop color by adding 150 ul of 1 mg ml chlorophenol VKM110902 Genlantis Page 2 of 3 Phone 858 457 1919 888 428 0558 U S Toll free e Fax 858 623 9494 www genlantis com red B D galactopyranoside CPRG Boehringer Mannheim dissolved in B gal buffer 1 mM MgCl2 10 mM KCI 50 mM B mercaptoethanol and 60 mM NazHPOs pH 8 0 Allow the reaction to proceed at room temperature until the red color develops 2 min to 4 hours depending on cell type Read absorbance at 580 nm An immunohistochemical approach for quantifying B galactosidase has also been reported Green Fluorescent Protein When green fluorescent protein GFP is the reporter gene used for transfections use epifluorescence or confocal microscopy to detect expression GFP has an excitation peak at 470 to 490 nm and emission peak at 510 nm Expression levels of GFP can also be monitored by fluorescence activated cell sorter analysis FACS
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