Transfection Protocol Calculator
Understanding Transfection Calculations
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Whether you are using lipofection, calcium phosphate precipitation, or electroporation, accurate scaling of DNA and reagent volumes is critical for experimental reproducibility and cell viability.
The Importance of DNA to Reagent Ratios
Most commercial transfection reagents (like Lipofectamine or FuGENE) recommend a specific ratio of reagent (in microliters) to DNA (in micrograms). Common ratios range from 2:1 to 4:1. Optimization is key; too little reagent results in low efficiency, while too much can be cytotoxic to your cell culture.
DNA Volume (µL) = Total DNA / Concentration
Reagent Volume (µL) = Total DNA × Ratio
Step-by-Step Protocol Example
Suppose you are performing a transfection in a 24-well plate (12 wells total) using 0.5 µg of plasmid DNA per well. Your stock DNA is at 1.0 µg/µL and you are using a 3:1 ratio.
- Step 1: Calculate total DNA. 12 wells × 0.5 µg = 6 µg. Adding a 10% overage for pipetting loss makes it 6.6 µg.
- Step 2: Determine DNA volume. 6.6 µg ÷ 1.0 µg/µL = 6.6 µL of DNA stock.
- Step 3: Determine reagent volume. 6.6 µg × 3 = 19.8 µL of transfection reagent.
Best Practices for High Efficiency
Always prepare a "Master Mix" for multiple wells to minimize pipetting variance. Ensure your DNA is of high purity (A260/A280 > 1.8) and free of endotoxins. Furthermore, cells should typically be 70-90% confluent at the time of transfection for optimal results in standard lines like HEK293 or HeLa.