Tips from the Bench: Transfection Tip
Optimize DNA Transfection
Cellular Behavior and Response Varies with Passage Number
Maintain a similar passage number between experiments to ensure reproducibility. A low passage number can make cells more sensitive to transfection whereas a high passage number can render cells refractory to transfection.
Use healthy, actively dividing cells to maximize transfection efficiency. Mirus Bio recommends plating cells the night before a transfection experiment at a density that will promote cell division to obtain 50-70% confluency for transfection the following day.
Use High Quality DNA for Transfections
Contaminants, such as protein, carbohydrate and lipids, may affect transfection efficiency and gene expression levels. Ensure that the plasmid preparation exhibits an A260/A280 ratio of > 1.8. Traces of contaminating endotoxin can be removed using Mirus Bio’s MiraCLEAN® Endotoxin Removal Kit.
Use an Optimal Volume of Transfection Reagent
When working with a new cell type, test a range of reagent volumes at a fixed DNA amount to find the level that achieves the highest transfection efficiency with minimal toxicity. For example, vary the concentration of Trans
IT®-LT1 Reagent from 2–8 μl per 1 μg DNA to find the optimal ratio.
Complex Formation Time
After mixing the DNA and transfection reagent, incubate to form complexes for 15-30 minutes at room temperature, before adding the mix to your cells. Transfection efficiency may decrease if the complex formation exceeds an hour.
Post-transfection Incubation Time
Depending on the gene being expressed and the experimental design, post-transfection incubation time can have a dramatic effect on experimental outcome. Protein expression is typically detectable as early as 4 hours post-transfection and can persist for many days. In general, maximal protein expression occurs 48 hours post-transfection. The time point for optimal gene expression can be determined by varying post-transfection incubation times from 4 to 72 hours.
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