Values given are for a 12-well plate. Values for different sized plates are listed in table I.
- Plate cells on a 12-well plate at an appropriate density so that they will reach 40-50% confluence the next day.
- Mix 4 µl of TransPass R1 siRNA transfection reagent (NEB #M2551S) in 100 µl of serum-free medium (e.g., High Glucose DMEM) in a sterile tube and mix by vortexing. Incubate at room temperature for 10-20 minutes.
- Add 1 µl of siRNA Mix1 to the diluted transfection reagent, mix gently by pipetting and incubate 10-20 minutes at room temperature to form the transfection complexes.
1 The concentration of siRNA Mix is 10 µM = 144.6 ng /µl.
- Dilute the complex with 500 µl of complete culture medium (e.g., DMEM, 10% FBS). (The final concentration of siRNA will be 15 nM).
- Aspirate the culture medium from the cells and immediately replace with the diluted transfection complex. Evenly disperse the siRNA complexes by gently rocking the plate.
Typical cell incubation time points for detecting target gene inhibition are 24-48 hours for mRNA and 48-72 hours for protein.