HomeTransfection & Gene EditingCo-transfection of Plasmid DNA

Co-transfection of Plasmid DNA

Georg Hannig & Cordula Jany

Roche Diagnostics GmbH


Figure 1.Successful co-expression of eGFP and mKate2. Fluorescent images of HeLa cells transfected with PC3.1eGFP alone (A) and pmKate2 alone (B), and co-transfection of the two plasmids using X-tremeGENE HP Reagent. Coexpression of eGFP and mKate2 is also shown for X-tremeGENE 9 Reagent (D). In Figure 1E, transfection efficiency of X-tremeGENE HP Transfection Reagents was calculated using the ratio of the area covered with fluorescing cells compared to the total area covered with cells. Indicated ratio of plasmids and reagent volume refers to 100 μL of transfection complex.

Transient co-transfection of plasmids is a method that is commonly employed for cellular protein-protein interaction studies, transcription factor studies, and gene knockdown studies using shRNA encoding plasmids. Thus, co-transfection is useful for a broad scientific community. In the present study, X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents (Roche) were tested for their co-transfection efficiency using two autofluorescent reporter genes, enhanced green fluorescent protein (eGFP), and red fluorescent protein (mKate2).

Transfection and automated imaging

HeLa Cells (ATCC) were plated at a density of 1.2 x 105 cells/100 μL per 96 well. The cells were transfected with PC3.1eGFP and pmKate2 plasmids (Evrogen) 24 hours after cell seeding. Three different pmKate2/PC3.1eGFP ratios were titrated (0.5 + 1.0, 0.5 + 0.5, 1.0 + 0.5 μg plasmid per 100 μL complex). In addition, four different amounts of X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents, respectively, were tested for complex formation (1, 2, 3, or 4 μL reagent per 100 μL complex). Transfection efficiency was measured 48 hours post transfection using the Cellavista Analyzer and HOECHST 33342 staining.


  • Transfection efficiencies with • two plasmids vary depending on the different ratios of the plasmids themselves and the relation of whole DNA/transfection reagent (Figures 1, A, B, and E).
  • Both X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents showed a high co-efficiency (Figure 1, C and D).
  • X-tremeGENE HP Transfection Reagent showed a more consistent transfection efficiency over all ratios in the tested HeLa cells.

For life science research use only. Not for use in diagnostic procedures.

X-tremeGENE is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

Hannig G, Jany C. 2013. Co-transfection of Plasmid DNA. BioTechniques. 54(1):
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