Lipofection-mediated DNA transfection assay
Lipofection-mediated DNA transfection assay
The following method is a modification of one studied by Mark Evans (Alexion Pharmaceuticals, New Haven, Connecticut). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Lipofection-mediated DNA transfection assay
Materials and Instruments
Exponentially growing mammalian cell cultures Move makings For more product details, please visit Aladdin Scientific website.
Lipid staining reagents NaCl sodium citrate plasmid DNA purified closed-loop plasmid DNA cell growth medium
Test tubes Polystyrene or polypropylene test tubes Tissue culture dishes
Buffers & Solutions
The composition of the storage solutions, buffers and reagents is shown in Appendix 1. dilute the storage solution to the appropriate concentration.
Lipid Dyeing Reagents 
NaCl (5 mol/L) (optional)
Used to dilute DOGA.
Sodium citrate (pH 5.5,20 mmol/L) containing 150 mmol/L NaCl (optional)
DOGS can be used in place of sterilized water for dilution of plasmid DNA when used as a lipid staining reagent (Kichler et al. 1998).
Nucleic acids and oligonucleotides
Plasmid DNA
If this is the first time you are lipofecting or using an unfamiliar cell line, you should use an expression plasmid that encodes either E. coli β-glucuronidase or green fluorescent protein (see Green Fluorescent Protein in the information column in Chapter 17). These plasmids are available from several manufacturers (e.g., pCMV-SPORT-β-gal,Life Technologies, pEGFP-F, CLOTECH; see Figures 16-2,16-3).
Purification of closed-loop plasmid DNA can be accomplished by chromatography columns or by ethidium bromide-CsCl gradient centrifugation as described in Chapter 1.
Dissolve the DNA in water to 1ug/ul. 
Culture medium
Cell growth medium [complete medium, serum-free medium and (alternatively) selective medium].
Specialty Equipment
Test tubes, polystyrene or polypropylene tubes
DGTMA binds non-specifically to polypropylene and polystyrene tubes must be used.
Tissue Culture Dish (60 mm)
This method is designed for use with 60 mm cell culture dishes. If using multi-well plates, cell vials, or flat dishes of different diameters, vary the cell density proportionally to the amount of specimen used. See Table 16-3.
Additional Reagents
Step 9 of this method may require reagents listed in Chapter 17, Protocol 7.
Cells and Tissues
Exponentially growing mammalian cell cultures 
Methods
1. 24 h before lipofection, collect cells by trypsin digestion and spread the cells on 60 mm tissue culture dishes (or 5X104 cells/35 mm dish) at a density of 105 cells/ dish. Add 5 ml of growth medium (3 ml for 35 mm dish) and incubate for 20-24 h at 37°C with 5%~7% CO2.
Cells should be 75% full grown at the time of transfection. If the cell growth is less than 12 h before transfection, the cells will not be well adsorbed on the culture dish and will be easily dislodged when they come into contact with lipids.
2. Dilute 1~10ug of plasmid DNA in a polystyrene or polypropylene tube with 100ul of sterilized deionized water (in the case of Lipofectin) or 20 mmol/L sodium citrate (pH 5.5) containing 150 mmol/L NaCl (in the case of Transfectam). In another tube, dilute 2~50ul of lipid solution to 100ul with sterilized deionized water or 300 mmol/L NaCl.
IMPORTANT: When transfecting with Lipofectin, use polystyrene tubes instead of polypropylene tubes because the cationic lipid DOTMA binds non-isotopically to polypropylene. For other cationic lipids, select tubes according to the manufacturer's recommendations.
3. Incubate for 10 min at room temperature.
4. Add the lipid solution to the DNA, mix by pipetting several times, and incubate for 10 min at room temperature.
5. Incubate the DNA-lipid solution by washing the transfected cells three times with serum-free medium, then add 0.5 ml of serum-free medium to each 60-mm dish, and incubate the cells in a 37°C incubator with 5% to 7% CO2 .
It is important to wash the cells to be transfected with serum-free medium before adding the DNA-lipid solution. Sometimes serum strongly inhibits transfection (Felgner and Holm 1989), as do extracellular matrix complexes such as proteoglycan sulfate, which may prevent the plasma membrane of the receptor cell from interacting with it by binding to DNA-lipids.
6. After incubating the DNA-lipid solution for 10 min, add 900ul of serum-free medium to each tube. Blow several times with a pipette to mix well and incubate for 10 min at room temperature.
7. Transfer the DNA-lipid solution from each tube to the cells in a 60 mm dish and incubate for 1~24 h in a 37°C incubator with 5%~7% CO2.
8. After the cells have been incubated in DNA for an appropriate period of time, wash them three times with serum-free medium and add complete medium to destabilize them.
9. If the cells are stably transfected, proceed to step 10. 24 to 96 h after lipofection, check the cells by one of the following methods.
- If plasmid DNA expressing β-glucuronidase is used, assay for enzyme activity in the cell lysate according to the procedure described in Chapter 17, Scheme 7, or by histochemical staining as described in Additional Scheme: Identification of β-glucuronidase by monolayer histochemical staining.
- If plasmid DNA expressing green fluorescent protein is used, examine the cells under a microscope at 450-490 nm.
- If other gene products are used as markers, newly synthesized proteins can be analyzed by in vivo metabolic markers such as radioimmunoassay, immunoblotting, immunoprecipitation, or analysis of enzyme activity in cell extracts.
To minimize differences in transfection efficiency between different culture bloods, it is best to (1). Transfect several culture dishes with each construct; (2). Digest the cells with trypsin after 24 h of incubation; (3). Pool the cells; and (4). Respread the cells on several petri dishes.
10. Isolation of stable transfectants: After 48-72 h of incubation in complete medium, trypsin digest the cells and resurface the cells with appropriate selection medium. The medium is changed every 2~4 days and the culture is continued for 2~3 weeks with the aim of removing dead cell debris and promoting the growth of resistant cells. In this way, independent clones can be cloned and propagated for assaying (see Jakoby and Pastan 1979 or Spector et al. 1998b [Chapter 86 of the Handbook of Cellular Chemistry] for methods).
Cells are fixed in pre-cooled methanol for l5 min, then stained with 10% Giemsa for 15 min at room temperature, rinsed under running water, so that the number of clones can be recorded.The Giemsa staining solution should be freshly prepared in phosphate buffer or water prior to use, and filtered through Whatman No. 1 filter paper. 