The following protocol for GeneFECTOR (TM) is applicable to all of our reagents.
OPTIMIZATION OF PARAMETERS: The conditions for optimal transfection efficiency vary between different cell types. To achieve the highest possible transfection efficiencies, several parameters need to be optimized. Once these parameters have been established for a particular cell line, reasonable reproducibility can be obtain from experiment to experiment. The following are the most important parameters:
Cell Confluence: Cell confluence should be between 50-70%, and kept as constant as possible from experiment to experiment. In general, it is easier to control this parameter in medium to large wells (6 well plates, 35-60 mm culture dishes) than in smaller sizes.
DNA/Liposome Complex: The optimal ratio of DNA/liposome and the total amount of DNA/liposome complex should be determined using a constant number of cells to obtain the highest transfection efficiency. This is easily achieved starting with a constant amount of DNA (for example, 1 µg per 35 mm dish) and varying the amount of GeneFECTOR (1-6 µg per dish in 0.5 or 1.0 µg increments). Then, keeping constant the DNA/liposome ratio which gave the highest efficiency in the previous experiment, vary the total amount of DNA/liposome complex and determine the new peak of activity. For example, if the DNA/liposome ratio in the first experiment was 1 µg/1 µg, then one could use 0.5 µg/0.5 µg, 1.0 µg/1.0 µg, 1.5 µg/1.5 µg and 2.0 µg/2.0 µg total amounts of DNA/liposome complex in the second experiment.
Transfection Time: Transfection efficiency is related to the exposure time of the cells to the DNA/liposome complex. In general, the longer the exposure time the higher the efficiency. However, since transfection is carried out in serum free or reduced serum medium, excessively long exposure times could lead to cell detachment or death. The recommended starting transfection time is 6-8 hour, however longer exposure times could prove optimal.
DNA TRANSFECTION OF ADHERENT CELLS (general protocol).
Cell plating: The following protocol is for 35mm dishes. For other sizes, adjust the volumes proportionally. The cells are plated in six 35mm dishes at a concentration such that their confluency will be 50-70% when transfected (usually 24-48 hours after plating).
GeneFECTOR:DNA complex: The DNA/liposome complex should be prepared at room temperature.
Note 1: Unsiliconized tubes can be used, but siliconized tubes appear to give better results
Note 2: The DNA: liposome complex MUST be made in serum free medium, otherwise negatively charged macromolecules in the serum will compete with the DNA for the liposome
Note 3: If the cells require serum at all times for survival , 0.8 ml of MEM with reduced (20-50% of normal) serum, but without antibiotics, can be used instead. Antibiotics present during transfection can kill the cells and can decrease the transfection efficiency.
Note 4: For serum free human keratinocytes, no FBS addition is needed. However, rinsing with medium containing BSA (0.5%) can be done to remove excess lipid/DNA complex. Then, serum free medium is added to restore the normal culture conditions.
DNA TRANSFECTION OF SUSPENSION CELLS. The following protocol is for 35 mm culture wells or dishes, and has been shown to work well with Jurkat cells.
Preparation of cells.
GeneFECTOR:DNA complex: Prepare the DNA/liposome complexes using the protocol described for adherent cells.
* Patent pending. For transgenic use, a signed agreement is necessary.