Protocole

GENERAL OPERATING PROCEDURE FOR CELL TRANSFECTION WITH PTG1

Transfection reagent:

Solubilize PTG1 at 1mg/mL in sterile 10 mM Hepes buffer, pH7.4. The solution can be aliquoted and stored at -20°C.

*Transfection HEK293

The day before the transfection

The cells (2x 105 in 1 ml culture medium) are seeded in 12-well culture plates.

The day of the transfection

Preparation of the transfecting solutions:

It is recommended to perform the transfection at DNA/PTG1 weight ratios of 1/3 and 1/6

For 3 wells of a 12-wells culture plate:

1) Transfection at DNA/PTG1 weight ratio of 1/3

– Add 15µL of PTG1 in 35 µL 10 mM Hepes buffer pH 7.4 containing 5 µg plasmid DNA

– Mix by 3 up-down pipettings  

– Vortex for 4s and keep for 30 min at room temperature.

– Complete to 1.5 mL with serum-free culture medium

2) Transfection at DNA/PTG1 weight ratio of 1/6

– Add 30µL of PTG1 in 70 µL 10 mM Hepes buffer pH 7.4 containing 5 µg plasmid DNA

– Mix by 3 up-down pipettings  

– Vortex for 4s and keep for 30 min at room temperature.

– Complete to 1.5 mL with serum-free culture medium

*Transfection CHO cells

The day before the transfection

The cells (2x 105 in 1 ml culture medium) are seeded in 12-well culture plates.

The day of the transfection

Preparation of the transfecting solutions:

It is recommended to perform the transfection at DNA/PTG1 weight ratios of 1/8 and 1/10

For 3 wells of a 12-wells culture plate:

1) Transfection at DNA/PTG1 weight ratio of 1/8

– Add 40µL of PTG1 in 90 µL 10 mM Hepes buffer pH 7.4 containing 5 µg plasmid DNA

– Mix by 3 up-down pipettings 

– Vortex for 4s and keep for 30 min at room temperature.

– Complete to 1.5 mL with serum-free culture medium

2) Transfection at DNA/PTG1 weight ratio of 1/10

– Add 50µL of PTG1 in 115 µL 10 mM Hepes buffer pH 7.4 containing 5 µg plasmid DNA

– Mix by 3 up-down pipettings 

– Vortex for 4s and keep for 30 min at room temperature.

– Complete to 1.5 mL with serum-free culture medium

Transfection

On the day of the transfection, the culture medium is removed and 500 μL of the transfecting solution are added to each well and the cells are incubated at 37°C. After 4 h, the medium is removed, replaced by 1 mL of complete culture medium (with 10% serum) and the cells are further cultured at 37°C. Gene expression is measured after 24h or 48h.

*Remark: This is a general protocol which could be refined (in particular the DNA /PTG1 Plus weight ratio) following the first transfections results and depending on the type of cells.