autoclaving tubes and buffers (again!) - (Aug/23/2010 )

Sorry, I know this has has been covered a bit before....

I'm about to start a new experiment that's a bit more finicky than what I usually work with, so I'm getting a bit nervous about avoiding contamination. Basically, I'm debating whether I want to autoclave my tubes and buffers. I don't really care about RNases since I'm just doing DNA work, but I do care about DNA contamination (whether bacterial or otherwise). Normally, I would just autoclave and be done with it, but the autoclaves in our building are also used for bacterial waste and soil samples, so who knows what kind of junk is hanging out in there. There are two things that I'm debating about:

1) Tubes: The tubes we buy are all certified DNase, RNAse, DNA and pyrogen free. They're not listed as sterile, but I don't see how they could be non-sterile and still be DNA free.

2) Buffer: I have to make a Tris/NaCl buffer for part of the protocol. All of the stocks going into the buffer are sterile, DNase and RNase free. If I just pipette them with sterile falcon pipets into a sterile bottle, do I really need to autoclave the whole solution afterwards? I'm worried about both contamination and evaporation here.

So, should I or shouldn't I?

Thanks!

You definitely do not need to autoclave the tips or the tubes if they are certified DNase and RNase free. If you are really concerned, you could use a fresh bag of tips and/or a new box of filter tips.

IF your stocks are sterile, and the water being used for dilution is also sterile and DNase and RNase free, then you can pipette into a sterile container (perhaps a 50 ml tube) and use the resulting solution without autoclaving.

DNA is largely broken down by autoclaving IIRC.

Two things: "DNAse free" does not mean "DNA free", and if you're concerned about the sterility of your buffers, but want to avoid autoclaving them, filter them through a 0.2 micron bottle-top filter.