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  • #16
    Hi Phillip,

    Thanks for your reply and I understand what you're saying about the different aspects of contaminants. We just haven't experienced difficulties with this, so I have been wondering about the validity of what I have heard about RNAse issues from autoclaving. As I said above, we use autoclaved tubes (and other stuff) in our RNA extractions and do many of them and get very good quality RNA. I think that Ethanol is correct on the point that RNAses are everywhere (more in the autoclave than in the lab, your hands, etc?) and that most will come from the sample itself.

    And, again, our culture media hasn't had a problem in the same autoclave that has been used for the 9 years I have been in our lab so if any enzymes or whatever are making their way into (or onto) our vessels, it isn't making a difference.

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    • #17
      I think what EthanOL was saying was that the RNAses mainly came from the samples themselves. Skin will probably have a little RNAse on it, but compared to the amount of RNAse in the cells that were homogenized to create the RNA prep? Probably not much. (Well, unless you just made a batch of 10 mg/ml RNAseA and spilled some...)

      If everything is going well for 9 years you will want to keep your own council on what is best way to go. Indeed, just because the autoclaves I have used at Purdue are filthy doesn't mean the ones used by your lab will be.

      --
      Phillip

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      • #18
        I think the word "contaminates" also depends on what experimentation you're performing. For example, I used to work at a university where we used the same autoclave for our waste as well as tubes,tips, etc. After autoclaving, the clear 1.7ml tubes would have a film on them that could be wiped off with your fingers. What was the film? Who knows? It was surely in every experiment used with those tubes. Whether it affected the results or not depends on what experiment you'd perform. For TC no one would have a problem because the junk on the tubes and tips was sterile. Yet, I had a very sensitive PCR assay for bacterial speciation using oligos designed to ultra conserved regions of the ribosome. I could take water that would not give me a PCR product and autoclave it. After, the water would be severely contaminated with bacterial DNA. Anything used for this assay (tubes/tips/water/buffer) had to be from virgin material and not in contact with anything from the autoclave.

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        • #19
          Originally posted by james hadfield View Post
          buy tubes from Ambion, AM12475. RNAse free non-stick. This is what we use.
          Just don't throw them in the autoclave because they'll be sticky and dirty afterwards. People go through all this crazy stuff to avoid RNase contamination and it is really difficult to introduce exogenous RNase into samples. I use to use massive amounts of RNase for RNase protection assays and I can tell you the amount of RNA degradation was always cell line dependent, which says the RNase is in the sample not coming from exogenous sources. You can go through all the song and dance you want, bathe in RNase ZAP but the real problem is DNA contamination. Which is worsened as most people routinely PCR the targets they are interested in. One femtogram of a 100 bp PCR product has the contaminating potential of 30 micrograms of genomic DNA in regards to your target of interest.

          On the autoclave issue, I think the best sense can be made by this story. Back in my graduate school days at Cold Spring Harbor Lab, there was some really good fishing just outside the lab in the late summer/early fall. Sometimes while I had a gel running or something I would drop a line in the water. One time when I was really hungry, I just couldn't wait until I finished up my experiments to go home and cook the fish, so I threw it in the autoclave and had a nice dinner out of it in the lab. Most people would think that was disgusting. Why, because the autoclave is aerosolizing all sorts of nasty junk caked all over the autoclave into my food (which was well wrapped in aluminum foil). They're probably right which is why your tubes are 'dirty' but sterile when they come out of the autoclave.

          Bottom line, your stuff is more dirty when it comes out of the autoclave. Will it have RNase on it? Unlikely. Enough human/mouse DNA to mess up your experiments? Unlikely. If you are looking at highly conserved bacterial sequences like 'brooksma', it will ruin you. The autoclave certainly isn't pushing things in the direction you want. So why do something that takes time and planing when it certainly isn't helping you and perhaps making things worse?
          --------------
          Ethan

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          • #20
            Anyone mentioned already that most of the standard tubes factory sealed in bags are essentially sterile? if you do not buy any fancy-treatment tubes that are further processed (siliconized or whatever) you should be on the safe side considering a fresh bag sterile. and i furthermore think that there is less RNAase and DNAse in them as compared to tubes that have been exposed in any way openly to your bench neighborhood, your hands even if gloved and of course the autoclave.

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