TranslationBlocker Human mTOR siRNA – DISCONTINUED

DISCONTINUED: Human mTOR TranslationBlocker™ siRNA Duplex Product Attributes

1. 1. Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. For instance, seed 12 well plates with 5x10⁴ cells in growth media and incubate at 37°C overnight before starting transfections. Transfection reagents may specify the desired level of confluence to look for prior to starting transfections.
   2. The above data was generated with Oligofectamine as a transfection agent in accordance with manufacturer’s instructions. Prepare transfection reagent in serum-free growth medium such as serum-free RPMI. Allow to come to room temperature. </ li>
   3. Dilute siRNA to working concentration in serum-free growth media. For Oligofectamine or similar transfections into 6 well plates, we recommend starting siRNA dose optimization at approximately 100 pmol siRNA duplex.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Ratio of transfection reagent to siRNA should be optimized. Equivalent volumes of prepared Oligofectamine or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 20 minutes with gentle mixing.
   5. Add prepared siRNA solution to cells. For 12 well plates this will entail adding the solution to cells in approximately 1.1 ml serum-free medium. Optionally, FITC labeled control siRNA (Product Number QC-1F) or positive control siRNA (Beta-Actin; Product Number QC-2) may be used to assess transfection efficiency.
   6. Incubate for several hours in serum free media. Generally, a 4 hour initial transfection incubation is recommended.
   7. Add serum containing media to culture without a media change if longer incubation times are required. Generally 1-2 fold of the original volume of siRNA containing serum-free media is added to restore nutrient levels in media. Alternatively, the media may be directly supplemented with serum such as 10% FBS.
   8. Incubate and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. mTor knockdown at the protein level was measured at 72 hours post transfection via Western Blot.