Establish stable cell clones

Transfection and drug selection

1. Transfect cells with a vector encoding a drug-selection gene (e.g., kanamycin resistance gene).

2. 36-48 hours after transfection, add drug selection agent (e.g. neomycin (G418) for vectors encoding kanamycin resistance gene).

3. Usually cells are harvested, diluted, and plated in fresh medium containing the slection agent.

4. The fold of dilution (1:1 to 1:100, commonly 1:5 to 1:10) depends on cell lines used and transfection efficiency. Clonies formed per 100 mm plate should be less than 100.

5. For suspension cell lines, cells may be plated into 96-well plates.

6. The drug concentration also varies depending on the cell line and the cell density. See also Establish stable cell clones --- drug concentrations.

7. Change to fresh medium containing the selection agent every 2-3 days.

8. Clonies should form 10-21 days for most fast growing cell lines.

Isolation of stable clonies

Protocol I

See Establish stable cell clones --- cloning cyclinders.

Protocol II

See Establish stable cell clones --- 96-well. FACS may be used to deliver single transfected cells into individual wells.

Protocol III

Mark the position of colonies. Wash the plate with PBS and add Trypsin-EDTA. Remove most Trypsin-EDTA before cells completely detach. Place a 1 ml pipet filled with 0.2 ml of medium directly on a colony (don't release the medium). Suck up and immediately transfer to a 48 or 24-well plate containing selecting agent.

Protocol IV

Wash the plate with PBS. Dilute Trypsin-EDTA 1:20 in warm PBS. Add to the plate. Find colonies on an inverted light (or fluorescence) microscope. Isolate using a pipette with a 20 ml tip, lower the tip to the surface of the colony and suck gently.