What "layers" are you referring to? while I am not an asp.net guru, the only layers (or tiers) that I would refer to when talking about web development in general is about the placement of components such as:
presenstation tier, business tier, and data tier. (typical 3 tier approach).
It's almost always a good idea to maintain a separation of concerns. It generally makes the troubleshooting process easier. If you need to change your data access you should be able to update your data access layer and not make changes to the rest of your application. The same he's for any other logical layers your application may include.