TransPass V: Transfection Protocol
Use Table 1 to select the reagent volumes for various plate sizes.
- Plate cells at an appropriate density so they will reach 70-80% confluence at the time of transfection (plating media should contain 10-20% serum)
- Mix plasmid DNA with serum-free DMEM (Table 1).
- Add the appropriate volume of HUVEC Reagent Component.
- Add the appropriate volume of TransPass V.
- Gently mix the transfection complex mixture by flicking the tube.
- Incubate at room temperature for 20-30 minutes.
- Add the transfection complex mixture to cells (Do not remove the serum-containing medium). Rock the plate gently in order to evenly disperse the transfection complex mixture.
- Return the plate to the incubator and incubate 24-48 hours before assay.
Note: The recommended ratio of HUVEC Reagent Component to TransPass V is between 1:1 and 1:2.
Table 1: DNA Transfection using TransPass HUVEC Transfection Reagent in different plate sizes
Culture Vessel Volume of Plating Medium (per well) DNA in serum-free medium volume HUVEC Reagent component in transfection complex mixture* TransPass V in transfection complex mixture* 96 well 100 µl 0.2 µg in 25 µl 0.1-0.2 µl 0.1-0.4 µl 24 well 500 µl 0.8 µg in 50 µl 0.5-1 µl 0.5-2 µl 12 well 1 ml 1.6 µg in 150 µl 1-2 µl 1-4 µl 35 mm dish 2 ml 3 µg in 250 µl 4-6 µl 4-12 µl 6 well 2 ml 3 µg in 250 µl 4-6 µl 4-12 µl 60 mm dish 5 ml 6 µg in 0.5 ml 8-12 µl 8-24 ml 100 mm dish 15 ml 18 µg in 1.5 ml 16-25 µl 16-50 ml
* The transfection complex mixture is composed of plasmid DNA and TransPass components in serum-free medium. For example, for a 12-well format, transfection complex mixture consisting of 1.6 µg plasmid, 1 µl HUVEC Reagent Component, 1 µl TransPass V and 150 µl serum-free medium is added to a well containing cells in 1 ml of 10–20% serum containing medium.