Procedure 1. The day before transfection, seed 46 x 105 cells (depending on the cell type) per well of a 6-well plate in 2 ml appropriate growth medium containing serum and antibiotics.
Note: Make sure that cells are in good condition and are seeded at least 24 h before transfection. Cells should be 8090% confluent on the day of transfection. 2. Incubate cells under their normal growth conditions (generally 37C and 5% CO2). 3. On the day of transfection, dilute 4 l Enhancer R in Buffer EC-R. Add 2 g RNA (minimum RNA concentration 0.1 g/l) and mix by vortexing for 10 s. The final volume should be 100 l.
For example, if the RNA concentration is 0.5 g/l, dilute 4 l Enhancer R in 92 l Buffer EC-R, then add 4 l RNA solution.
IMPORTANT:
Always mix Enhancer R with Buffer EC-R before adding RNA.
Always keep the ratio of RNA to Enhancer R constant.
Note:
The best results are achieved when RNA of the highest purity is used for transfection. RNA purified with RNeasy Kits and Oligotex mRNA Kits is highly recommended. 4. Incubate at room temperature (1525C) for 5 min, then spin down the mixture for a few seconds to collect drops from the top of the tube. 5. Add 8 l TransMessenger Transfection Reagent to the RNAEnhancer R mixt
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