Sorry Stefania, but I have to disagree. Autoclave will only break the hydrogen bonds between strands, and they will re-anneal once the temperature drops. It will still amplify in PCR - autoclave is useful if you work with pathogens, but pretty useless (or even harmful as your contamination from a single piece of equipment can get spread around to your other stuff). You have other options if you want to make stuff DNA-free. Bleach is the most frequently used, cheap and works really well (we clean all lab benches daily with bleach). UV works, but only on exposed surfaces. Gamma radiation would work, but I'm not sure it's a viable option.

But I'm all with you on filter tips. Also, we do a complete separation of laboratories - DNA extraction and PCR setup are done in a lab where PCR products are never handled. With a lab where we handle noninvasive or ancient material (scats, hair, saliva, old bones etc.) we completely limit movement of people and material - if you go to a post-PCR lab, you can't go back until you've showered and changed your clothes. :)

Thanks Stefania, but does these DNA fragments contaminate or interfere in PCR amplification.

Sorry Stefania, but I have to disagree. Autoclave will only break the hydrogen bonds between strands, and they will re-anneal once the temperature drops. It will still amplify in PCR - autoclave is useful if you work with pathogens, but pretty useless (or even harmful as your contamination from a single piece of equipment can get spread around to your other stuff). You have other options if you want to make stuff DNA-free. Bleach is the most frequently used, cheap and works really well (we clean all lab benches daily with bleach). UV works, but only on exposed surfaces. Gamma radiation would work, but I'm not sure it's a viable option.

But I'm all with you on filter tips. Also, we do a complete separation of laboratories - DNA extraction and PCR setup are done in a lab where PCR products are never handled. With a lab where we handle noninvasive or ancient material (scats, hair, saliva, old bones etc.) we completely limit movement of people and material - if you go to a post-PCR lab, you can't go back until you've showered and changed your clothes. :)

Hmm... have to go back on that one. Went to take a look and it seems that autoclave does help, but you need to do it for a long time (2h+). I'm still not doing it in our lab - clean plastics, bleach and separation of laboratories seems a safer option.

Gefrides, Lisa A., et al. "UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables." Forensic science international: genetics 4.2 (2010): 89-94.

I really think bleach is the way to go for exposed surfaces if you want to get rid of possible contamination. It always worked for me, in addition to separating working areas of extraction and PCR. Always use filtered-tips and all the rest should be available in the kit, which is mostly DNA free :)

Best of luck.

I agree with Tomaz. Autoclaving will NOT remove all traces of DNA. An easy and sure way of decontaminating your glassware, instruments and surfaces (but slightly more expensive than bleach) is to use reagents especially made for that such ad "DNA away" ( http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152___11964348_-1 ) or "DNA Off" ( http://www.clontech.com/takara/US/Products/Molecular_Biology/Molecular_Biology_Reagents/General/DNA-OFF_and_RNase-OFF )

even after autoclaving the degraded DNA single strands will be there.on the pcr amplification, if the primer we are using is specific to any of these single stranded DNA contaminant the further procedures of pcr amplification will multiply those DNA fragments so even after autoclaving there can be chances of unwanted, unexpected DNA.autoclaving is not a good method for making the things free from DNA contamination.

Thanks all for your very informative inputs. I have one more ambiguity can we make bulk soil DNA free, if yes then how.

Hi praveen I donot have any idea what do you want to do but i can suggest you to use DEPC treated reagents in your work. This will completely remove the DNA and no contamination will occur. But you cannot use DEPC treated reagents in certain experiments. Best....

Gamma/X-ray radiation? Another question is how to get this capacity... not something you have in your average molecular biology lab. Got any friends at a local friendly nuclear power plant? ;o)

Hi, I would have used nucleases if it would have been less amount !! Since, you are talking of BULK then it is not feasible option !! It also depends upon your downstream application, for that matter virtually nothing is DNA free in any environmental sample :)

Acid treatment (10% acetic acid or hydrochloric acid) should hydrolyze destroy all DNA. Then do extensive rinsing with milliQ water. You should be able to treat your bulk soil with that too. Not sure what you want to do with your soil afterwards though.

I use acid treatment, followed by rinsing and 70% EtOH wash to clean all my electroporation cuvettes to get rid of all the crap/junk from previous electroporations.

Thanks all for nice suggestions. Yes acid treatment is good choice, I did this for making soil microbe free, but never think that it can make DNA-free also. Nucleases treatment will be difficult for bulk soil. I want to track microbes in microbe-free soil using molecular markers, so need microbe-free and DNA -free soil.

Actually, we were discussing the paper published in Microbial Ecology. "Changes in Soil Bacterial Community Structure with Increasing Disturbance Frequency. (DOI 10.1007/s00248-013-0237-9; also see attachment PDF).

Authors of the paper 'created' the 'disturbance' in soil sample by adding 'autoclaved' soil (claimed to be DNA free). Later they analysed the bacterial community abundance by NG sequencing. So, question were raised, whether added soil was DNA free. If not (consensus from discussion above) all results can be called futile!!?

Hi Praveen and Amit

As much as i understand for this purpose you can treat the soil with DEPC and autoclave the soil. this will help you to eradicate whatever Nucleic acid present in the Soil. You can also use the acid treatment as suggested.

cheers

Well as an author of that godawful Microbial Ecology paper, I think I'd better clarify something :) . We did consider the issue of DNA carryover but we relied on reassuring sources suggesting that DNA is reduced to levels too low to amplify. One more recent example is http://www.biotechniques.com/BiotechniquesJournal/2013/December/Decomposition-of-waste-DNA-with-extended-autoclaving-under-unsaturated-steam/biotechniques-348850.html

As we autoclaved at 121 C for 90 minutes, it exceeded the necessary. We SHOULD really have tested this ourselves, but we took it as a given. Next time we will test it for ourselves!

Anyway, if you look at the bacterial community ordination of the different disturbance regimens, none of those is anywhere close to the 'original' soil community that was DNA extracted just before autoclaving. So it doesn't look like that original DNA is dominating the system.

We now tried extracting DNA from soil that had been autoclaved under the said conditions (as 1.5 kg in autoclave bag). Extractable amount of DNA using a Mobio kit was very small, and failed to give any amplification band upon PCR (16s, ITS1). This really shows that soil autoclaved like this is 'clean' for practical purposes.