CHO Cell transfection

(from RC 8/99)

Day before

Split cells 1:2 to give a confluent plate the next day. (Cells are normally maintained in 15 cm dia. TC plates.)

Day of:

§ Aspirate medium and wash with 10 ml PBS

§ Add 2.5 ml of pre-warmed (37 deg) Trypsin-EDTA and rock gently for 3-5 min.

§ Add 10 ml of PBS and pipet up and down to remove cells.

§ Spin 1000 x g for 5 min.

§ Wash again in PBS and pellet at 1000 x g for 5 min.

§ Resuspend in Dulbecco’s: minus-Mg⁺⁺, minus-Ca⁺⁺ PBS. Use 1 ml per 15 cm dish of cells).

§ Pipet DNA into 0.4 cm gap cuvette (blue). (Determine amount of DNA empirically, try 10 ug at first.)

§ Add 0.5 ml of cell suspension to each cuvette and mix gently by pipeting up and down.

§ Elecroporate:

1 pulse @ 0.34 kV, 960 mF capacitance.

Time constant should be about 13 – 19 msec.

§ Add 1 ml of fresh medium to cuvette and suck off+ the cell-debris. Transfer the cells to 12 ml of fresh medium and aliquot 0.5 ml/well in a 24 well culture dish with sterile glass cover slips. ALTERNATIVELY, pipet 1 ml onto a sterile (+)-charged slide and place 4 slides into 15 cm plate. Surface tension will hold media on the slide.

§ After 4 - 8 hr, gently aspirate medium and add fresh medium.

§ After 18 hr, replace medium again.

§ Examine for expression 24 to 48 hr post-transfection.