y 37C and 5% CO2). 3. On the day of transfection, dilute 1.6 l Enhancer R in Buffer EC-R. Add 0.8 g siRNA (minimum siRNA concentration, 0.1 g/l) and mix by vortexing. The final volume should be 100 l.
IMPORTANT:
Always mix Enhancer R with Buffer EC-R before adding RNA. Always keep the ratio (g:l) of siRNA to Enhancer R constant (1:2). 4. Incubate at room temperature (1525C) for 5 min, and then centrifuge the mixture for a few seconds to collect drops from the top of the tube. 5. Add 4 l TransMessenger Transfection Reagent to the siRNAEnhancer R mixture. Mix by pipetting up and down 5 times, or vortexing for 10 s. 6. Incubate the samples for 10 min at room temperature (1525C) to allow transfectioncomplex formation. 7. While complex formation is taking place, gently aspirate the growth medium from the plate, and carefully wash cells once with sterile PBS using 1.52 times the volume of media used for cell seeding.
IMPORTANT:
Do not allow the cells to become dry. Minimize the time they are without medium. 8. Add 300 l cell growth medium (without serum or antibiotics) to the tube containing the siRNATransMessenger Reagent complexes. Mix by pipetting up and down twice, then immediately add the transfection complexes drop-wise onto the cells. Gently swirl the pla
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