Optimization of GenMute™ reagent for siRNA/DNA co-transfection.

GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations for siRNA/DNA co-transfection range from 0.5 nM to 10 nM.  Excessive siRNA may lead to "flooding effect" which may comprise silencing effect, so never use siRNA higher than 20 nM. The following procedures will guide to optimize GenMute™ reagent for best siRNA/DNA co-transfection in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute™ reagent.

1. Change medium and add 0.5 ml of complete medium to each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.
2. Dilute 0.5 µg DNA to each sterile tube of total 4 tubes with 50 µl of GenMute transfection buffer (Cat # SL100572) followed by addition series diluted siRNA to each well of the total 4 tubes-------add 5.0 pmol siRNA to 1st tube, 2.5 pmols to 2nd tube, 1.25 pmols to 3rd tube and 1.25 pmols to 4th tube. Let's sit at RT for 5 minutes.
3. Add 3.0 µl GenMute™ reagent to diluted siRNA/DNA of each tube, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.
4. For 4 consecutive wells of 24-well plate, add the transfection complexes which are prepared with same concentration of DNA (0.5 µg per well) and different 4 concentrations of siRNA ranging from final 10 nM to 1.25 nM.
5. Check silencing effect in the 4 wells 24~48 hours post transfection and choose the best transfection conditions.