1. Cat # SL100668 Store at 4 0 C LipoD293 DNA In Vitro Transfection Reagent (Ver. II) 100  l 500  l 1000  l 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 Toll Free. 1-(866)-918-6812 Email: info@signagenlabs.cominfo@signagenlabs.com Web: www.signagenlabs.comwww.signagenlabs.com Introduction: LipoD293™ (Ver. II) is an enhanced liposome-based DNA transfection reagent which is specifically formulated and optimized for HEK293 cells and other mammalian cells with superior efficiency and invisible cytotoxicity. LipoD293™ Reagent (Ver. II), 1.0 ml, is sufficient for 300 to 600 transfections in 24 well plates or 50 to 100 transfections in 6 well plates. Important Guidelines for Transfection: - While the following protocol is for 293 cells, follow the same procedures for other types of mammalian cells. To request protocols for lentivirus production and sf9 insect cell transfection, please email us at info@signagenlabs.com - For high efficiency, transfect cells at high density. ~90% confluency is highly recommended - To lower cytotoxicity, transfect cells in presence of serum (10%) and antibiotics Procedures for Transfecting Mammalian Cells 1. For Adherent Cells Cell Seeding (see Table 1): Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal ~90% confluency at the time of transfection. Complete culture medium with serum and antibiotics is freshly added to each well 30~60 minutes before transfection. Note: For high efficiency, high cell density (~90% confluency) is essential. High serum levels (>5%) usually do not have inhibitory effect on transfection efficiency. For some specific cells, maximal transfec- tion efficiencies are observed in presence of serum and antibiotics. We recommend using complete serum/antibiotics-containing medium initially. Table 1. A Guideline for Seeding mammalian Cells Prior to Transfection in Different Culture Formats This product is for laboratory research ONLY and not for diagnostic use  2008 SignaGen Laboratories Culture DishesSurface Area (cm2)Number of Cells to Seed T175 Flask1750.7–1.4 x 10 7 T75 Flask753.0–6.0 x 10 6 100 mm Dish582.2–4.4 x 10 6 60 mm Dish210.9–1.8 x 10 6 35 mm Dish9.63.5–7.0 x 10 5 6-well Plate9.64.0–8.0 x 10 5 12-well Plate3.51.5–3.0 x 10 5 24-well Plate1.90.8–1.6 x 10 5 48-well Plate1.04.0–8.0 x 10 4 96-well Plate0.31.2–2.4 x 10 4 Preparation of LipoD293™-DNA Complex and Transfection Procedures We recommend the LipoD293™ (L):DNA (g) ratio of 3:1 as a starting point which usually gives satisfactory transfection efficiency with invisible cytotoxicity. To ensure the optimal size of complex particles, we recommend using serum-free DMEM with High Glucose to dilute DNA and LipoD293™ Reagent. The following protocol is given for transfection in 24- well plates, refer to Table 2 for transfection in other culture formats. The optimal transfection conditions for a majority of adherent cell lines are given in the standard protocol described below. - For each well, add 0.5 ml of complete medium with serum and antibiotics freshly 30~60 minutes before transfection. - For each well, dilute 1 µg of DNA into 50 µl of serum-free DMEM with High Glucose. Vortex gently and spin down briefly. - For each well, dilute 3 µl of LipoD293™ reagent into 50 µl of serum-free DMEM with High Glucose. Vortex gently and spin down briefly. - Add the diluted LipoD293™ Reagent immediately to the diluted DNA solution all at once. (Important: do not mix the solutions in the reverse order !) - Vortex- mix the solution immediately and spin down briefly to bring drops to the bottom of the tube followed by incubation of 15~20 minutes at room temperature to allow DNA-LipoD293™ Reagent complexes to form. Note: Never keep the DNA/LipoD293™ complex longer than 20 minutes - Add the 100 µl LipoD293™/ DNA mixture drop-wise onto the medium in each well and homogenize the mixture by gently swirling the plate. - Remove LipoD293™/DNA complex-containing medium and replace with complete serum/antibiotics containing medium 12~18 hours post transfection. For sensitive cells, to lower cytotoxicity, remove LipoD293™/ DNA complex and replace with complete medium 5 hours after transfection. - Check transfection efficiency 24 to 48 hours post transfection. Storage: LipoD293™ DAN In Vitro Transfection Reagent (Ver. II) is stable for up to 12 months at 4 0 C. This item shipped at ambient temperature

2. Cat # SL100668 Store at 4 0 C LipoD293 DNA In Vitro Transfection Reagent (Ver. II) 100 l 500 l 1000 l 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 Toll Free. 1-(866)-918-6812 Email: info@signagenlabs.cominfo@signagenlabs.com Web: www.signagenlabs.comwww.signagenlabs.com This product is for laboratory research ONLY and not for diagnostic use  2008 SignaGen Laboratories 2. For Suspension Cells The following protocol is given for transfection in 24-well plate. The protocol can be scalded up or down according to the surface area of culture vessels. Cell Seeding: Suspension cells are typically seeded the day of the transfection at a density of 2 x 105 cells per well (24-well plate). For optimal transfection conditions with LipoD293™, seed the number of cells adapted to the culture vessel format according to Table 3. Table 2. Recommended Amounts for Different Culture Vessel Formats Culture DishTransfection Volume (ml) Plasmid DNA (g) Diluent Volume (mL) LipoD293™ Reagent (L) 96-well plate0.2 2 x 0.010.6 48-well plate0.30.52 x 0.021 24-well plate0.51.02 x 0.053 6-well plate1.022 x 0.16 35 mm dish1.022 x 0.16 60 mm dish2.852 x 0.2515 100 mm dish67 - 82 x 0.521 - 24 T75 flask818 - 362 x 0.7554 - 108 250 ml flask2050 - 1002 x 1.25150 - 300 Table 3. Recommended Number of Suspension Cells to Seed Culture DishNumber of Cells 96-well plate2 x 10 4 – 5 x 10 4 48-well plate5 x 10 4 – 1 x 10 5 24-well plate1 x 10 5 – 2 x 10 5 6-well plate2 x 10 5 - 5 x 10 5 35 mm dish5 x 10 5 – 2 x 10 6 60 mm dish2 x 10 6 – 5 x 10 6 100 mm dish5 x 10 6 – 1 x 10 7 LipoD293™/DNA Complex Preparation and Transfection procedures: For different cell types, the optimal ratio of LipoD293™ (L):DNA (g) varies from 1:1 to 3:1. We recommend the LipoD293™ (L):DNA (g) ratio of 3:1 as a starting point which usually gives satisfactory transfection efficiency with invisible cytotoxicity. To ensure the optimal size of complex particles, we recommend using serum-free DMEM with High Glucose to dilute DNA and LipoD293™ reagent. - The day before transfection, plating cells in 24- well plates at 2x10 5 cells per well so that they are ~90% confluent on the day of transfection. Cells are plated in 0.5 ml of their normal growth medium containing serum and without antibiotics. Note: High serum levels (>5%) usually do not have inhibitory effect on transfection efficiency. Sometimes growth medium without serum and antibiotics gives the best efficiency. We recommend replacing normal serum-containing medium with serum-free and antibiotics-free medium 30 minutes before transfection. - For each well, dilute 1 µg of DNA into 50 µl of DMEM Serum-free Medium with High Glucose. - Vortex gently and spin down briefly. - For each well, dilute 3 µl of LipoD293™ reagent into 50 µl of serum-free DMEM Medium with High Glucose. Vortex gently and spin down briefly. - Add the diluted LipoD293™ Reagent to the diluted DNA solution all at once. Note: Do not mix the solutions in the reverse order! - Vortex- mix the solution immediately and spin down briefly to bring drops to the bottom of the tube followed by incubation of 15 minutes at room temperature to allow DNA-LipoD293 Reagent complexes to form. - Add the DNA-LipoD293 Reagent complexes (100 µl) directly to each well and mix gently by rocking the plate back and forth. - Incubate the cells at 37°C in a CO 2 incubator for 24 ~ 48 hours until they are ready to assay for transgene expression. Alternatively, growth medium may be replaced after 5 hours without loss in transfection activity. Note: For transfection performed in serum-free medium, be sure to replace with normal growth medium (with serum and antibiotics) after 5 hours incubation! Scale-up: In general, for larger size plates, scale up all amounts, reagents, and volumes in proportion to the surface area. Some final optimization can then be made as to what is optimal for the larger dish size.