TransPass V: Transfection Protocol

Overview

Use Table 1 to select the reagent volumes for various plate sizes.

Protocol

1. 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)
2. Mix plasmid DNA with serum-free DMEM (Table 1).
3. Add the appropriate volume of HUVEC Reagent Component.
4. Add the appropriate volume of TransPass V.
5. Gently mix the transfection complex mixture by flicking the tube.
6. Incubate at room temperature for 20-30 minutes.
7. 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.
8. 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.