Protocol of pcDNA3.1-GFP Transfection Cells

The liposome transfection method refers to the positively charged surface of cationic liposomes, which can be with the phosphate of nucleic acids through electrostatic interaction of DNA molecules wrapped into the formation of DNA a lipid complex, by the surface of the negatively charged cell membrane adsorption, and then through the fusion of the membrane, and occasionally through the direct penetration of the role of the DNA delivery into the cell, the formation of inclusion bodies or into the lysosomes A small portion of the DNA can be released from the inclusion body and into the cytoplasm, and further into the nucleus of the transcription, expression.

• The day before transfection, cells are trypsin digested and counted, and cells are plated so that their density is 90% on the day of transfection. Cells are plated in 0.5 mL of normal growth medium containing serum and no antibiotics.
• For each well of cells, dilute 0.8 μg-1.0 μg of DNA using 50 μL of serum-free DMEM medium. Batch preparation is possible for multi-well operation.
• For each well, dilute 1-3 μL of LIPOFECTAMINE 2000 Reagent with 50 μL of DMEM medium. After dilution with LIPOFECTAMINE 2000, mix with the diluted DNA within 5 minutes. Prolonged holding time reduces activity.
• Mix diluted DNA (from step 2) and diluted LIPOFECTAMINE 2000 (from step 3). Hold at room temperature for 20 minutes.
• Add the complex directly to each well, shake the plate, and mix gently.
• Hold at 37°C in 5% CO₂ for 24-48 hours without removing the complex changing the medium or changing the growth medium after 4-5 hours without decreasing the transfection activity.
• Add the complex to the cells.
• After 24-72 hours of adding the complex to the cells, analyze cell extracts or perform in situ cell staining to detect reporter gene activity. This is dependent on cell type and promoter activity. For stable expression, cells are passaged into a fresh medium one day after starting transfection, and screening antibiotics are added two days later. Several days or weeks are required to perform stable expression.

• Transfection is not possible with a culture medium containing serum, which has a great impact on transfection efficiency.
• Use a double-antibody-free medium when spreading plates before transfection, the increase in resistance will affect the transfection efficiency.
• Transfection is performed within 24 hours of plate laying.
• Be gentle throughout the operation and avoid violent shaking.

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