plastics for RNA

[mimila]

Hello everyone,

I have a problem with tubes which I recently have bought from Bioplastics. There is information Dnase Rnase and pirogen fre but also on the label is "non-sterile". I want to isolate RNA and I am afraid of losing expensive reagents for isolation because of tubes. Is it possible that non-sterile tubes are Rnase and Dnase free? I do not understand it. What should I do?
Thanks in advance

Marta

[grassgirl]

What kind of tubes (microfuge, etc.) are they? Can they be autoclaved? If so, they will be sterile once autoclaved.

[mimila]

I have 0,5 1,5 and 2 ml tubes
They all are autoclavable, I wonder if it is necessary to autoclave it?
If there are not Dnases and Rnases there should not be any organisms, so why they are not sterile and what contaminations are possible?

[grassgirl]

I would say the the necessity to autoclave depends on how clean the tubes have been kept. Autoclaving won't get rid of RNAses, and if you think about it your tissue isn't sterile either. But if lots of people have been getting into a supply of tubes, you may consider autoclaving if you want them to be clean. However, some people claim that you can actually introduce RNAses by autoclaving because of killing, for instance, bacteria and having RNAses released, which are not destroyed by the autoclave.

If it were me I would try a test extraction or two with the tubes not autoclaved and see how yield and integrity are.

[mimila]

I also have read about introducing Rnases after autoclaving and I think that theory is quite logical. Taken into account that autoclaving does not destroy Rnases I think that parameters :non-sterile and Rnase Dnase free together are very stupid. I think it is impossible. You have wright that only way is to try it but I have no reagents for that knd of testing. I have only kit for few reactions for mRNA purification and this step is very important for me, wrrrrrrrrrrrr

[grassgirl]

Well, another idea is to get some new tubes that are sterile AND nuclease free. Seems like a less expensive route than using up kit reagents. Perhaps you can get some free samples from a vendor...often times this is the case

Good luck!

[mimila]

I wonder if it is typical to sell non-sterile Rnase free tubes? I changed the company from Axygen and I have not nice suprice now with Bioplastics. Unfortunately I have no time for new order now:/
Have you ever tried this kind of tubes, or always sterile?

[grassgirl]

Actually, for the most part I get tubes similar to yours and autoclave them out of the bag simply because we, as a rule, autoclave all our microfuge tubes in the lab. I do get a new autoclaved set of tubes when I do RNA work so that RNAses haven't been introduced by opening and closing the supply (personally I worry more about this than the possibility from autoclaving). I've had very good results from RNA extractions, but there are many more things to consider than the tubes you're putting it in (a VERY clean bench, clean gloves changed frequently, clean bench, aerosol pipet tips, keeping your extractions on ice, etc.). I also store the RNA in a buffer that contains RNAse inhibitors in the case of any leftover RNAses. If you take the strategy that RNAses are simply everywhere and do a good job of minimizing them, I think that works pretty well.

Hopefully this hasn't confused the issue even more.

[james hadfield]

buy tubes from Ambion, AM12475. RNAse free non-stick. This is what we use.

[brooksma]

I, as a rule, NEVER autoclave any of the tubes I am using for molecular biology applications...especially RNA work. Autoclaving can introduce many unwanted things to tubes including RNases. There is no point to autoclave tubes for RNA work, sterility is only required for tissue culture applications. Non-sterile tubes mean that the tubes haven't been gamma irradiated before sale...so what? They are RNase-free which is what counts. Just observe good RNA practice when using the tubes and you'll be fine.

[pmiguel]

Yes, have you ever taken a look at the inside of an autoclave? If it is anything like ones I have seen, they are filthy. Typically because of boil-over from various sources--including bacterial trash. On top of that, steam is typically adulterated with chemicals to reduce the amount of scale they leave on pipes. (No idea why the steam has dissolved solutes in it, but apparently it does.)

--
Phillip

[ETHANol]

Listen to the last two posts.

First the paranoia around RNase contamination is completely unfounded. Yes, RNase is a problem but it comes from your sample itself. You have to have pretty bad lab practices to introduce exogenous RNase into you samples. When you see protocols that tell you to DEPC treat a solution be very suspicious because it is not only unnecessary but can cause problems. DNA contamination is a much bigger threat to experiments but gets a tenth the attention.

As far as autoclaving, I beat my head against the wall trying to get students and postdocs to understand that autoclaving will make your tubes, tips, etc. MORE dirty and does nothing to reduce RNase/DNA contamination. You don't have to imagine it, you can actually see the filth on things that have been autoclaved with your naked eyes. Think about it, would you pipet a few microliters of that goop in the autoclave try into your solutions after you autoclave. You might as well because that stuff is aerosolized into them.

Cell culture - Autoclave
Molecular Biology/Biochemistry - Do NOT autoclave

[protist]

I agree with ETHANol. We never autoclave tubes for molecular biology - aside from the potential issues mentioned in previous posts autoclaving weakens the tubes. Our tubes of choice for libraries are 2 mL Sorenson™ Dolphin microcentrifuge tubes - excellent for very small volumes.

[grassgirl]

You don't have to imagine it, you can actually see the filth on things that have been autoclaved with your naked eyes. Think about it, would you pipet a few microliters of that goop in the autoclave try into your solutions after you autoclave. You might as well because that stuff is aerosolized into them.

Cell culture - Autoclave
Molecular Biology/Biochemistry - Do NOT autoclave

I would like to, respectfully, ask you to explain this logic. We autoclave bacterial culture waste in the same autoclave as we use for new cell culture media (and there is no goop left, by the way...everyone uses trays). Autoclaving kills the bacteria, so no bacteria should be alive in your newly autoclaved media, even if it could enter the vessel the media are autoclaved in (we use small Erlenmeyers with foil on top). If you are worried about contaminants in the autoclave, how can you autoclave your cell culture stuff and be sure it's sterile/non-contaminated? Further, are you saying that contaminants permeate the closed container that is used for autoclaving (for microtubes we use beakers, again covered with double heavy-duty foil).

The tube damage idea makes sense to me, but not the introduction of contaminants. Just wondering.

[pmiguel]

Contaminants are not all equal. Bacteria are complex and can usually be killed by autoclaving. (Although this presumes you are autoclaving long enough to bring the volume of liquid to the desired temperature for a long enough period of time.)

But you can kill the bacteria and still have intact (active) enzymes left behind. Further lots of autoclave trash will be replete with RNAse A.

Ideally everyone using the autoclave is very careful to make sure their autoclave trash does not boil over into the autoclave. Or, if there is a spill, they clean it up. I have yet to see an autoclave in this "ideal" state a month after it is installed.

--
Phillip