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
- 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.
- 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.
- Incubate at room temperature for 15 minutes.
- Add 1-3 µg of plasmid DNA and/or 1.5-30 pM of siRNA to the reagent-medium mixture.
- Incubate at room temperature for 15 minutes.
- Add the transfection complex mixture to the well, and evenly disperse the complex mixture by gently rocking the plate.
- Return the plate to the incubator and incubate cells overnight.
- 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
VessselSurface
(cm2)Volume of
Plating Medium
(per well)TransPass R1
in Serum-free
MediumDNA in
Transfection
Complex MixturesiRNA
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