Where to sample from? Does it matter?

I've just started brewing again for the first time in nearly 15 years. When I brewed before I only used buckets for the primary and was pretty much blind to what was happening. Now that I'm using carboys I can watch the process (which is awesome). I'm noticing that in my HG brews there are noticeable layers in the fermenter which change often. I'm not about to agitate it to get a homogeneous mixture, but does it really matter where you take a sample from for a SG reading?

Not really IMO, I usually try to grab the sample from the middle of the liquid. I have to put the thief in twice to get enough. I don't really think that it would make a whole lot of difference if I grabbed it form the bottom though.