GenMuteTM siRNA轉染試劑（Cat#SL100568）是市場上最有力的siRNA傳遞工具之一。siRNA/DNA共轉染時，siRNA的最佳濃度范圍是0.5ηM到10ηM，過多的siRNA可能導致沉默效果差的“洪水效應”，切勿使用超過20ηM的siRNA。以24孔板為例，如下步驟將對如何優化GenMuteTM轉染試劑做一指導；至于其他規格器皿的培養的細胞，煩請參考GenMuteTM的實驗說明。
1、在轉染前的30-60分鐘，更換培養基，并在每孔中加入0.5ml的完全培養基（含血清和抗生素）。
2、將0.5μg DNA各自加入到分別盛有50μl GenMute轉染緩沖液（Cat#SL100572）的4個無菌管中進行稀釋，接下來分別對4管中的siRNA進行系列稀釋·····將5.0pmol siRNA加入到第一管中，2.5 pmol siRNA加入到第二管中，1.25 pmol siRNA加入到第三管中，1.25 pmol siRNA加入到第四管中。室溫下靜置5分鐘。
3、取3.0μl GenMuteTM轉染試劑，分別加入到稀釋好的siRNA/DNA管中，混勻，室溫下靜置15分鐘以形成轉染復合物。請注意：室溫下，轉染復合物的靜置時間勿超過25分鐘。
4、將如上制備的轉染復合物分別加入24孔板中連續的4個孔中。
5、轉染24-48小時后，分別檢測4孔中的沉默效果并選擇出最佳的轉染條件。
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.