|Transfection in the presence of serum || |
- Serum, once thought to decrease transfection efficiency, can be present during transfection as long as the DNA-cationic lipid reagent complexes are formed in the absence of serum. Some serum proteins interfere with complex formation.
- The optimal amounts of cationic lipid reagent and DNA may change in the presence of serum; thus optimize conditions with serum if you plan to add it to the transfection medium.
- Most cells remain healthy for several hours in a serum-free medium.
|Antibiotics in the culture medium|| |
- Cationic lipid reagents increase cell permeability. This increases the amount of antibiotics delivered into the cells and results in cytotoxicity. Lower transfection efficiency may result. Therefore do not use antibiotics in the transfection medium.
- Avoid using antibiotics when plating cells for transfection. This reduces the need for rinsing the cells before transfection.
- For stable transfections, do not use penicillin and streptomycin in selective medium because the antibiotics are competitive inhibitors of Geneticin® selective antibiotic.
- When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.
- If using serum-free medium, use lower amounts of antibiotics than you would in serum-containing medium to maintain the health of the cells.
|Cell maintenance and evolution of cultures|| |
- For optimal transfection results, follow a routine sub-culturing procedure.
- Passage cultures once or twice a week at a dilution that allows them to become nearly confluent before the next passage.
- Do not allow the cells to remain confluent for more than 24 hr.
- Cell cultures evolve over months and years in the laboratory, resulting in changes in cell behavior with regard to transfection. Thawing a fresh ampoule of cells allows recovery of transfection activity.
|Cell plating density || |
- The optimal cell density for transfection varies for different cell types or applications. For adherent cells, generally 70% to 90% confluency at the time of transfection or 5 × 105 to 2 × 106 suspension cells/ml provides good results.
- Make sure cells are not confluent or in stationary phase at the time of transfection.
- Since transfection efficiency is sensitive to culture confluence, maintain a standard seeding protocol from experiment to experiment.
- In some cases, an increase in the number of cells plated increases the transfection activity.
|DNA quality|| |
- High-quality, intact plasmid DNA is important for achieving high performance transfections.
- Invitrogen recently has launched a line of nucleic acid purification kits called PureLink™ HiPure Purification Kits (Mini, Midi, Maxi, Mega, and Giga) that provide high quality DNA for transfections. Log on to www.invitrogen.com/nap for more information.
- Cesium chloride banding yields highly purified DNA but is labor intensive and time consuming.