It pains me the funniest single joke I've seen in MST3K history was at a live show that wasn't recorded so there's no evidence of it.
During a climactic battle, the protagonist's weapon runs dry and he throws it out of frame down a corridor, out of frame.
Tom shouted "Hallway, cover me!"
Close second: Cry Wilderness' running joke of a lot of adult characters having guns on them and the kid character keeps surprising them, so one of the bots or Noah would shout "BANG" at a time it'd be hilarious if they accidentally shot him.
