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In my first play-through of Alchemical Lab, I accidentally placed two towers on either side of a chest spawn, a bit too close.

I found that I could not open the chest as a result. Fortunately, selling the towers (iirc I had to sell both) allowed me to open it.

Is it possible for a poorly-placed defense to prevent a chest from spawning altogether, or will I always be able to sell the defense to open the chest should I make this mistake again in the future?

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up vote 7 down vote accepted

In the Alchemical Laboratory, I have a tendency to plop a Slice n' Dice right where I marked it on the map here in red, on top of the chest spawn. I've found that it easily takes care of all the enemies spawning from the East door. The chest has always spawned, even with my tower right on top of the spawn area.

As long as you are able to touch the chest, you should be able to open it. Sometimes I had to shimmy around my tower a little, but I was always able to access the chest.

Alchemical Lab

Based on this experience, I would say that no, placing a tower on top of a chest spawn will not prevent it from spawning, it will just make it more difficult to open.

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After looking at your map, I realized I had the wrong level. That's exactly the chest I had wedged underneath two cannonball towers! – Beofett Oct 25 '11 at 16:01

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