From 2007.igem.org

When the cells in the flask are between 80% and 95% confluence:

• Remove all of the old medium.
• Wash with approx 3 ml of PCB.
• Add 4 ml of Trypsin; rinse for 30 seconds.
• Remove all but 0.5 ml of trypsin and let sit for two minutes.
• Add 5 ml of new medium. Verify that all cells are being detached from the surface of the flask.
• Once all cells are in suspension, pipette out a desired fraction of the medium. The amount removed depends on the purpose to which the cells will be put. If the passaging is just to stop the flask from being over crowded then removing 5/6ths of the cells is often appropriate. (Note that the amount removed along with the doubling time determine how often the passaging needs to be done.)
• Return flask to carbon dioxide incubator.