TransPass R1: Transfection Protocol

Overview

The amounts below are given for a 12-well plate format. Use Table 1 to adjust the reagent volumes for other plate sizes.

Protocol

1. Plate cells in complete growth medium containing 10% serum and no antibiotics/antimycotics at an appropriate density, so that they will reach 70-80% cell density (plasmid transfection) or 40-60% (siRNA alone or plasmid and siRNA transfection) at the time of transfection.
2. Add 4 µl of TransPass R1 Transfection Reagent to 100 µl of serum-free medium (e.g., DMEM/high glucose) in a sterile tube (one per sample well) and mix well.
3. Incubate at room temperature for 15 minutes.
4. Add 1-3 µg of plasmid DNA and/or 1.5-30 pM of siRNA to the reagent-medium mixture.
5. Incubate at room temperature for 15 minutes.
6. Add the transfection complex mixture to the well, and evenly disperse the complex mixture by gently rocking the plate.
7. Return the plate to the incubator and incubate cells overnight.
8. Replace the transfection medium with complete medium (i.e. 10% FBS-DMEM) the next day and incubate 24-72 hours before assaying. 
   
   Table 1: Plasmid DNA transfection in the presence of serum
   

   Culture Vesssel	Surface (cm2)	Volume of Plating Medium (per well)	TransPass R1 in Serum-free Medium	DNA in Transfection Complex Mixture	siRNA Concentration in Final Volume*
   96 well	0.32	75 μl	0.1 μl in 10 μl	0.1 μg	2.5-50 nM
   48 well	0.95	125 μl	1.5 μl in 25 μl	0.3 μg	2.5-50 nM
   24 well	1.9	250 μl	2.5 μl in 50 μl	0.7 μg	2.5-50 nM
   12 well	3.8	500 μl	4 μl in 100 μl	1-3 μg	2.5-50 nM
   6 well	9.5	1 ml	6-9 μl in 250 μl	3 μg	2.5-50 nM
   60 mm dish	21	2 ml	15-20 μl in 500 μl	6 μg	2.5-50 nM
   100 mm dish	55	7 ml	40-100 μl in 1 ml	15-20 μg	2.5-50 nM

   *Final volume =  plating medium plus the transfection complex mixture composed of TranPass R1 Transfection Reagent and plasmid DNA/siRNA in serum free medium.