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0C Cat # SL100668 Store at 4 LipoD293 DNA In Vitro Transfection Reagent (Ver. II) 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 100 l Toll Free. 1-(866)-918-6812 500 l Email: firstname.lastname@example.org 1000 l Web: www.signagenlabs.com This product is for laboratory research ONLY and not for diagnostic use Preparation of LipoD293™-DNA Complex and Introduction: Transfection Procedures LipoD293™ (Ver. II) is an enhanced liposome-based DNA We recommend the LipoD293™ (L):DNA (g) transfection reagent which is specifically formulated and ratio of 3:1 as a starting point which usually optimized for HEK293 cells and other mammalian cells with gives satisfactory transfection efficiency with superior efficiency and invisible cytotoxicity. LipoD293™ invisible cytotoxicity. To ensure the optimal size Reagent (Ver. II), 1.0 ml, is sufficient for 300 to 600 of complex particles, we recommend using transfections in 24 well plates or 50 to 100 transfections in serum-free DMEM with High Glucose to dilute 6 well plates. DNA and LipoD293™ Reagent. Important Guidelines for Transfection: The following protocol is given for transfection in 24- - While the following protocol is for 293 cells, follow the well plates, refer to Table 2 for transfection in other same procedures for other types of mammalian cells. To culture formats. The optimal transfection conditions request protocols for lentivirus production and sf9 insect for a majority of adherent cell lines are given in cell transfection, please email us at email@example.com the standard protocol described below. - For high efficiency, transfect cells at high density. ~90% - For each well, add 0.5 ml of complete medium with confluency is highly recommended serum and antibiotics freshly 30~60 minutes before - To lower cytotoxicity, transfect cells in presence of serum transfection. (10%) and antibiotics - For each well, dilute 1 µg of DNA into 50 µl of Procedures for Transfecting Mammalian Cells serum-free DMEM with High Glucose. Vortex gently 1. For Adherent Cells and spin down briefly. Cell Seeding (see Table 1): - For each well, dilute 3 µl of LipoD293™ reagent Cells should be plated 18 to 24 hours prior to transfection so into 50 µl of serum-free DMEM with High Glucose. that the monolayer cell density reaches to the optimal Vortex gently and spin down briefly. ~90% confluency at the time of transfection. Complete - Add the diluted LipoD293™ Reagent immediately culture medium with serum and antibiotics is freshly added to the diluted DNA solution all at once. (Important: do to each well 30~60 minutes before transfection. not mix the solutions in the reverse order !) Note: For high efficiency, high cell density (~90% - Vortex- mix the solution immediately and spin down confluency) is essential. High serum levels (>5%) briefly to bring drops to the bottom of the tube usually do not have inhibitory effect on transfection followed by incubation of 15~20 minutes at room efficiency. For some specific cells, maximal transfec- temperature to allow DNA-LipoD293™ Reagent tion efficiencies are observed in presence of serum complexes to form. and antibiotics. We recommend using complete Note: Never keep the DNA/LipoD293™ complex longer serum/antibiotics-containing medium initially. than 20 minutes - Add the 100 µl LipoD293™/ DNA mixture drop-wise Table 1. A Guideline for Seeding mammalian Cells onto the medium in each well and homogenize the Prior to Transfection in Different Culture Formats mixture by gently swirling the plate. Culture Dishes Surface Area (cm2) Number of Cells to Seed - Remove LipoD293™/DNA complex-containing medium T175 Flask 175 0.7–1.4 x 107 and replace with complete serum/antibiotics containing 2008 SignaGen Laboratories T75 Flask 75 3.0–6.0 x 106 medium 12~18 hours post transfection. For sensitive 100 mm Dish 58 2.2–4.4 x 106 cells, to lower cytotoxicity, remove LipoD293™/ 60 mm Dish 21 0.9–1.8 x 106 DNA complex and replace with complete medium 5 35 mm Dish 9.6 3.5–7.0 x 105 hours after transfection. 6-well Plate 9.6 4.0–8.0 x 105 - Check transfection efficiency 24 to 48 hours post 12-well Plate 3.5 1.5–3.0 x 105 transfection. 24-well Plate 1.9 0.8–1.6 x 105 Storage: LipoD293™ DAN In Vitro Transfection Reagent 48-well Plate 1.0 4.0–8.0 x 104 (Ver. II) is stable for up to 12 months at 4 0C. This item 96-well Plate 0.3 1.2–2.4 x 104 shipped at ambient temperature 0C Cat # SL100668 Store at 4 LipoD293 DNA In Vitro Transfection Reagent (Ver. II) 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 100 l Toll Free. 1-(866)-918-6812 500 l Email: firstname.lastname@example.org 1000 l Web: www.signagenlabs.com This product is for laboratory research ONLY and not for diagnostic use 2. For Suspension Cells The following protocol is given for transfection in 24-well plate. The - The day before transfection, plating cells in 24- protocol can be scalded up or down according to the surface area of well plates at 2x105 cells per well so that they culture vessels. are Cell Seeding: ~90% confluent on the day of transfection. Cells Suspension cells are typically seeded the day of the transfection at a are plated in 0.5 ml of their normal growth density of 2 x 105 cells per well (24-well plate). For optimal medium containing serum and without antibiotics. transfection conditions with LipoD293™, seed the number of cells Note: High serum levels (>5%) usually do not adapted to the culture vessel format according to Table 3. have inhibitory effect on transfection efficiency. Sometimes growth medium Table 2. Recommended Amounts for Different Culture Vessel without serum and antibiotics gives the Formats best efficiency. We recommend replacing Culture Dish Transfection Plasmid Diluent LipoD293™ normal serum-containing medium with Volume (ml) DNA (g) Volume (mL) Reagent (L) serum-free and antibiotics-free medium 30 96-well plate 0.2 0.2 2 x 0.01 0.6 minutes before transfection. - For each well, dilute 1 µg of DNA into 50 µl of 48-well plate 0.3 0.5 2 x 0.02 1 DMEM Serum-free Medium with High Glucose. 24-well plate 0.5 1.0 2 x 0.05 3 - Vortex gently and spin down briefly. 6-well plate 1.0 2 2 x 0.1 6 - For each well, dilute 3 µl of LipoD293™ reagent into 50 µl of serum-free DMEM Medium with High 35 mm dish 1.0 2 2 x 0.1 6 Glucose. Vortex gently and spin down briefly. 60 mm dish 2.8 5 2 x 0.25 15 - Add the diluted LipoD293™ Reagent to the diluted DNA solution all at once. 100 mm dish 6 7-8 2 x 0.5 21 - 24 Note: Do not mix the solutions in the reverse T75 flask 8 18 - 36 2 x 0.75 54 - 108 order! 250 ml flask 20 50 - 100 2 x 1.25 150 - 300 - Vortex- mix the solution immediately and spin down briefly to bring drops to the bottom of the Table 3. Recommended Number of Suspension Cells to Seed tube followed by incubation of 15 minutes at room temperature to allow DNA-LipoD293 Culture Dish Number of Cells Reagent complexes to form. 96-well plate 2 x 104 – 5 x 104 - Add the DNA-LipoD293 Reagent complexes (100 48-well plate 5 x 104 – 1 x 105 µl) directly to each well and mix gently by rocking the plate back and forth. 24-well plate 1 x 105 – 2 x 105 - Incubate the cells at 37°C in a CO2 incubator 6-well plate 2 x 105 - 5 x 105 for 24 ~ 48 hours until they are ready to assay for transgene expression. Alternatively, growth 35 mm dish 5 x 105 – 2 x 106 medium may be replaced after 5 hours without 2008 SignaGen Laboratories 60 mm dish 2 x 106 – 5 x 106 loss in transfection activity. 100 mm dish 5 x 106 – 1 x 107 Note: For transfection performed in serum-free medium, be sure to replace with normal LipoD293™/DNA Complex Preparation and Transfection growth medium (with serum and procedures: For different cell types, the optimal ratio of antibiotics) after 5 hours incubation! Scale-up: LipoD293™ (L):DNA (g) varies from 1:1 to 3:1. We In general, for larger size plates, scale up all recommend the LipoD293™ (L):DNA (g) ratio of 3:1 as a amounts, reagents, and volumes in proportion to starting point which usually gives satisfactory transfection the surface area. Some final optimization can efficiency with invisible cytotoxicity. To ensure the optimal size then be made as to what is optimal for the of complex particles, we recommend using serum-free DMEM larger dish size. with High Glucose to dilute DNA and LipoD293™ reagent.