TranslationBlocker Rat Apoptosis regulator Bcl-2 siRNA – DISCONTINUED

DISCONTINUED: Rat Apoptosis regulator Bcl-2 TranslationBlocker™ siRNA Duplex Product Attributes

Apoptosis regulator Bcl-2 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed 6 well plates with 1×10⁵ cells and incubate at 37°C for 24 hours.
   2. Prepare transfection reagent in serum-free growth media such as Opti-MEM allow to come to room temperature. The above data was generated with Lipofectamine 2000 for delivery of Rat Bcl-2 siRNA prepared in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For Lipofectamine 2000 or similar transfections into 6 well plates, we recommend starting siRNA concentration optimization at around 200 pmol siRNA duplex in 150 ul of serum free media. Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared Lipofectamine 2000 or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 30 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells. In this case 300 ul of siRNA complexed with Lipofectamine 2000 was added to 2.5 ml serum-free media. Additionally, 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.
   7. Optional: Add serum containing media to culture without a media change. Generally 1-2 fold of the original volume of siRNA containing serum free media is added to restore nutrient levels in media.
   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. Rat Bcl-2 knockdown at the protein level was measured at 120 hours post transfection via western blot. Additional studies suggest Bcl-2 knockdown may occur as soon as 24 hours post-transfection.