If the main sentence is an imperative sentence, such as,
"Do it at once,"
Grammatically speaking, should the tag question that follows the imperative sentence be "will you?" or "won't you?" not "don't you?"
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I disagree with the accepted answer. Tags are actually fairly commonly used with the imperative mood to make it a stronger command or request. Both negative and positive tags can appear with imperative sentences, and what form the tag question is used in depends on the context and generates nuances in meaning. In certain contexts a positive tag following an imperative is preferred as it introduces another layer of forcefulness and authoritativeness. It is heard very often in informal speech.
Leave me alone, will you? (Examples of this also abound on Google Books counting among them: work by renowned poet Charles Bukowski, book by Claudia Carroll, and oh you can't forget the legendary Vladimir Nabokov's classic Lolita can you now?)
I think at this point I don't have to continue listing examples from Google Books, as obviously there are a plethora of them. Reinforcing an imperative with a positive tag is very common in colloquial English.
Now as for the sentence at issue. Ending it with different tags brings about different subtleties.
Do it at once, will you not?
This sounds slightly formal and also a bit dated.
Do it at once, won't you?
This sounds like the speaker wants to confirm the volition of the listener and the recipient of the demand with the tag question.
Do it at once, will you?
This is slightly informal in register but also comes with a definitive and authoritative tone. A very likely subtext: "I expect you to do it at once."
Apart from these tags, you also have a multitude of other options: "could you?" "can't you" "would you?" "why don't you?" "why can't you?" I won't go into much details with each one of these.
Do it at once, could/would you?
Some people like to make demands with "class". It is still an order but softened and toned down with a polite "could/would you".