Place it at the turning point of a reaction sequence, after the setup line and before the fallout beat. Follow it with a bruh soundboard button for a deadpan aftermath, or lead in with an oh my god meme sound button to build the disbelief first.
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This is a half-second clip of the GoAnimate Catherine voice delivering a single, flat 'No!' with no extra words around it. The bluntness is the joke, giving listeners an instant sense of rejection, disbelief and awkward finality. It comes from the Catherine text-to-speech voice used in those animated videos.