Time from the first one rotating. Allow sufficient roll time for the second and clear it for takeoff. eg 747 rotates at 10:00:00, the next is a DH8C which only takes 15-20 seconds from brakes off to rotate, so you might say "cleared for takeoff" at 10:01:40.
A 757 might take 25-30 seconds from brakes off, but if it's positioning and light then it might only be 15-20 seconds. You get a feel for this after a while.
Although you can mess about with a few seconds here and there for slots / MDIs etc, you absolutely can't with vortex as it is such a safety issue. Therefore it's better to be a few seconds over the required time, but the closer you can be to the exact 2 minutes, the more expeditious and efficient you are.