Cloning cylinders

1. Mark well separated clones on the bottom of the tissue culture plate with a marker pen. Select clones that are well separated from the others and at least 100 cells.

2. Remove the growth media from the plate and rinse the cells once with PBS.

3. Place a cloning cylinder over a colony of cells so that the cells are in the center of the cylinder using a forceps. Gently press the cylinder to the plate. Forceps and cloning cylinder should have been thoroughly cleaned and sterilized by autoclaving.

4. Add 20-50 ml Trypsin-EDTA to dissociate cells.

5. As soon as cells have detached, transfer cells to 96 or 48-well plate using a pipette tip. Fill the pipette with 50-100 ml growth medium, carefully add to the Trypsin-EDTA in the cloning cylinder, and them remove cells.

6. Add enough FCS containing medium to inactivate Trypsin-EDTA. If the medium contains little or no serum, remove Trypsin-EDTA in the step 4 before cells detach.