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Transient Transfection FAQs part2

2018-11-29 Hits(759)

Q2: Reduced cell viability following transfection:

Possible cause(s)	Suggested solutions
Antibacterial agents were used in growth medium during transfection.	Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
Transfection reagent stored improperly.	We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
Complexes not thoroughly mixed in growth medium.	Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA: transfection reagent complexes in the wells.
Cells have changed over time, or splitting conditions have changed.	If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
Cationic lipid reagent was oxidized.	Do not vortex or agitate cationic lipid reagents excessively, this may form cationic lipid reagent peroxides.
Selection antibiotic added too soon.	When creating stable cell lines, allow at least 72 h for cells to express the resistance gene before adding selective antibiotic.

Q3: Transfection results not reproducible：

Possible cause(s)	Suggested solutions
Cells have changed over time, or splitting conditions have changed.	If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
Transfections performed at different cell confluencies, or at different DNA: transfection reagent ratios.	Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA: transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.