Transfection Basics Support—Troubleshooting
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Here are possible causes for reduced viability following transfection, along with suggested solutions. Please note that as per recent findings, antibiotics can be used in media during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.
Please review these possible causes for non-reproducible transfections, along with suggested solutions. Also, we recommend setting up one master mix of the DNA/lipid complex for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats.
A common reason for seeing precipitate on your cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance.
We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon® Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells.
You may also consider using a viral-based system to deliver your gene into your mammalian cell line of interest.
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