I was always against autocalving plastic before use them for PCR or RNA Extraktion. Open the box from the supplier only with gloves in a clean surrounding (bench cleaned with H2O2) was enough in my opinion. PCR tube were only cleaned by exposure to UV-light.
Here they autoclave things for PCR once and for RNA extraktion even twice to get them really clean.
Now I'm struggeling: Which is the right way? How do you handle it?
And am I right that RNase refolds itself after high temperatures?
Hi Karin,
I think it depends on the 'grade' of your plastic ware. If the supplier states its RNAse-, DNAse-, pyrogen-free and so on, I wouldn't autoclave it. For other non-gamma-rayed stuff it might be worth it. For example, we autoclave the standard bag tips for cloning work. BTW we checked the heat degradation of DNA for our digital PCR work flow (Jahn et al., Anal Chem, 2014) and DNA is rapidly degraded at 95°C if not protected by special buffer. In the case of enzymes like RNAses I don't know the heat sensitivity, but in my former lab they baked all RNA work stuff at 200°C for some time.
Hope that helps a bit?
Micha
At uni we always autoclaved things for molecular work.
Here we don't, and PCRs etc. still work.
so, I guess it's up to you=)
autoclaving things twice however, seems to be..unecessary, at least in my opinion.
I agree with Micha - I would always use PCR grade plastics for PCR and RNA and this means that autoclaving is not necessary or can even contaminate the plastics e.g. with spores if the autoclave is also used for waste inactivation. I have also learnt that baking at 200°C (we even did 220°C for 2 h) is the only safe thermal treatment for glass ware to get rid of any RNase activity. Admittedly, I never checked it, but I would suppose that RNases can survive twice autoclaving.
For PCR I expose tubes under UV while for managing RNA I usually put clean tubes in a glass box, cover it with alluminium and autoclave it for 20 minutes and once dried I put them under UV .... ^_^
Its easy to do it but if your plastic is not sterile you can do it under UV , with formalin , with ethanol......?
I autoclave everything for molecular work. If something is not working, it will be a painful process to figure out what's wrong. So, autoclaving gives me peace of mind.
I wonder how uv sterilization work: how are tubes sterilized when uv does not reach inside tubes? Plastic filters uv light so the uv does not penetrate inside. Will they be sterile?
On the other hand, gas sterilization will occupy all space, so it makes a sense.
For doing DNA stuff, I autoclave 0.5-ml, 1.5-ml and 2-ml microcentrifuge tubes (buy in bulk), and all pipet tips (1000 ul, 200 ul, and 10 ul). I don't do anything to the PCR tubes. For more than 10 years, it seems to work fine for me.
Whatever you do or don't do, just keep a negative control or non-template control in your experiments. That will directly solve your query as when and what to autoclave. Even we also do the same as Yuan does and we don't get any trouble. For sensitive assays use RNase-free, DNase-free, pyrogen-free, gamma sterile plasticwares which need not be autoclaved. As per my knowledge, RNase is not easy to degrade even if autoclaved. So, better open and use the materials in a clean sterile area.
Thanks everyone for your answers! It seems like there is no wrong or right way between the two I know.
Now I feel more confident and will establish my favored way of handling the plastic here.
For RNA work you need to more careful than working with DNA. It is best that bench and items related to handling should be clean with RNAse guard (commercially available). For your plastic such as eppendorf tubes, best toopen the box (obviously with gloved hands) and make small batches in plastic containers with screw lids and after autoclaving keep the containers closed and open only when needed. The idea is to keep tubes clean from dust. One of the thing that is very dangerous working with RNA is that no body is sneezing in the lab and around your bench. Sneezing is the best source of RNAse in the area.
I hope it helps.
As you have already realized after all these responses, there is not a clear consensus wether autoclaving is necessary for RNA work or not. I personally do not autoclave brand new plastics, and I'm getting beautiful RIN10 from my samples. But I'm scrupulously carefull with dust, and bare hands.
Just to add some documentation on that, here I attache two Ambion's (now Life Technologies, sorry) tech notes about the issue. I also add the links, just in case.
If you carefully read them you'll see that in the one they tell autoclaving is not efficient because of refolding, while in the other they provide proves that autoclaving actually inactivates RNase A.
They are anyways worth to read by novice (or not so much) in the RNA field.
http://www.lifetechnologies.com/es/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/rnase-and-depc-treatment.html
http://www.lifetechnologies.com/es/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/ten-sources-of-rnase-contamination.html
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