Cat # SL100488-HEPG2 Store at 4 0CGenJet In Vitro DNA Transfection Reagent for HepG2 Cells (Ver. II) ----- A Protocol for Transfecting HepG2 Cells 100 l 500 l 1000 l15875 Gaither DriveGaithersburg, MD 20877FAX. 301-560-4919TEL. 301-330-5966Toll Free. 1-(866)-918-6812Email: info@signagenlabs.comWeb: www.signagenlabs.com

Introduction:GenJet In Vitro DNA Tranfection Reagent (Ver. II) is upgraded version ofGenJet In Vitro DNA Tranfection Reagent. With a new chemistry, more DNA condensing groups were released in the new version compared with old version GenJet, leading to 3~4 times more efficient in DAN delivery. GenJet (Ver. II) for transfecting HepG2 cells was formulated for tanasfection of HepG2 cells. Procedures for Transfecting HepG2 Cells:Step A. Cell Seeding (see Table 1):Cells should be plated 18 to 24 hours prior to transfection so that themonolayer cell density reaches to the optimal ~90% confluency at the time of transfection. Complete culture medium with serum and anti-biotics is freshlyadded to each well ~60 minutes before transfection.Note: To obtain healthy HepG2 cells, the cells must be grown on a culture dish pre-treated with Collagen type I. Table 1. A Guideline for Seeding Adherent Cells Prior to Transfection in Different Culture FormatsThis product is for laboratory research ONLY and not for diagnostic use 2008 SignaGen LaboratoriesStep B. Preparation of GenJet-DNA Complex and Transfection ProceduresFor HePG2 cells, the optimal ratio of GenJet (L):DNA (g) is 3:1. We recommend the GenJet (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 GenJet 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 HepG2 cells are given in the standard protocol described below. - For each well, add 0.5 ml of complete medium with serum and antibiotics freshly ~60 minutes before transfection.- For each well, dilute 1 g of DNA into 50 l of serum-free DMEM with High Glucose. Vortex gently and spin down briefly to bring drops to bottom of the tube .- For each well, dilute 3 l of GenJet reagent (Ver. II) into 50 l of serum-free DMEM with High Glucose. Vortex gently and spin down briefly.- Add the diluted GenJet 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 bottom of the tube followed by incubation of 15~20 minutes at room temperature to allow GenJet-DNA complexes to form. Note: Never keep the DNA/GenJet complex longer than 20 minutes- Add the 100 l GenJet/ DNA complex drop-wise onto the medium in each well and homogenize the mixture by gently swirling the plate.- Remove DNA/GenJet complex-containing medium and replace with fresh complete serum/antibiotics containing medium 12~18 hours post transfection. - Check transfection efficiency 24 to 48 hours post transfection.

Storage: GenJet DAN In Vitro Transfection Reagent is stable for up to 12 months at +4 0C. This item shipped at ambient temperature

Table 2. Recommended Amounts for Different Culture Vessel Formats

Culture DishesSurface Area (cm2)Number of Cells to SeedT75 Flask753.0 6.0 x 106100 mm Dish582.2 4.4 x 10660 mm Dish210.9 1.8 x 10635 mm Dish9.63.5 7.0 x 1056-well Plate9.64.0 8.0 x 10512-well Plate3.51.5 3.0 x 10524-well Plate1.90.8 1.6 x 10548-well Plate1.04.0 8.0 x 10496-well Plate0.31.2 2.4 x 104

Culture DishTransfection Volume (ml)Plasmid DNA (g)Diluent Volume (mL)GenJet Reagent (L)96-well0.20.22 x 0.010.648-well 0.30.52 x 0.02124-well 0.51.02 x 0.0536-well 1.222 x 0.1635 mm dish 1.522 x 0.1660 mm dish352 x 0.251510 cm dish 67 - 82 x 0.521 - 24T75 flask1018 - 362 x 0.7554 - 108250 ml flask2050 - 1002 x 1.25150 - 300