293T Transfection

I. Split cells

remove DMEM(+) medium (with 10% serum and PS) from T25 flask;

Add 5 ml PBS to the flask to wash the cells;

remove PBS;

Add 0.5 ml 1X trypsin;

Keep at room temperature until the cells are loose (1~2 min)

Add 5 ml DMEM(+) to the flask, use pipette to disperse cells by pipetting liquid up and down.

Add 10 ml DMEM(+);

Transfer cells to 12 well plate, 1 ml/well, keep the last 3 ml, add 1 ml DMEM and continue to culture.

Add 3ul transfection reagent to 50 ul DMEM (-) (without serum and PS) medium;

Add 1 ug DNA to 50 ul DMEM(-) medium;

Transfer the transfection reagent to DNA and mix well;

Keep at room temperature for 15-20 min;

Add entire 100 ul mixture to 12 well plate prepare in step I, drop by drop;

Put the 12 well plate back to incubator.

--Ztao 10:41, 29 March 2011 (EST)