DNAse co-purifies with RNAse, many commercially available preps contain traces of DNAse. Fortunately RNAse is extremely thermostable. You can boil yor RNAse for 10 minutes to denature the DNAse and then centrifuge to pellet out any contaminants.

DNase contamination in any of your reagents (including the RNAase)? Or insufficient precipitation process? Lost pellet in the "dry samples in centrifuge" step?

Hi Berthold,

Thanks for the suggestions! The RNase A is supposed to be endonuclease-free (guaranteed by the manufacturer), and the remaning reagents are used during regular DNA extraction, so it's not apparent where the contamination would be. Also, while the vacuum centrifuge step has potential risk of losing pellets, this is usually not a problem in any other context. If my memory serves me, I think we once (long ago) saw the loss of DNA already after incubation with RNase A. I will ask my colleagues (who are in the lab while I'm on computer duty at the moment) to quantify already after the initial incubation.

Martin,

btw, a DNAse contamination test would be rather easy to do with any of the reagents - just incubate a bit of sound DNA with the reagents, and do a gel with that DNA before/after incubation.

Many years ago, I tried to isolate DNA from birch pollen - very unsuccessfully. In the end I found out that the pollen, once in contact with water, degrades added DNA very efficiently ...

So if any of you ever run out of DNase in the lab ... just use birch pollen :-)

Haha, that's a great and cheap substitute... Who would have thought birch pollen had such superpowers. Will suggest the simple DNase contamination test. Thanks again!

Been there, seen that.

Buy a fresh stock of Qiagen RNAse.

Good luck.

Hi, had the same problem of low DNA yield. Precipitation was, in my case not efficient enough, so I extended the "Let rest in freezer for 30min" step to "Let rest over night". That increased yield. Good luck!

DNAse co-purifies with RNAse, many commercially available preps contain traces of DNAse. Fortunately RNAse is extremely thermostable. You can boil yor RNAse for 10 minutes to denature the DNAse and then centrifuge to pellet out any contaminants.

Hi........I would like to know more details such as thermal stability of the DNA of your interest at 37 degrees for 30minutes because if the DNA is getting denatured while/after incubation, it's each strand is now susceptible to RNAse degradation..............have you checked the above situation by decreasing incubation time (here again you will face problem with RNAse activity that may decline)

May I know what is your plan to do with that DNA?

If you are going to run a PCR, you hardly get any trouble with that RNAse as it is going to degrade when u do PCR with initial degradation step itself i.e. 94 C.

By the way what was your sample volume, if you have enough carry again phenol:chloroform extract your DNA after the RNase digestion very carefully.

or

if your DNA is partly degraded I do not think it gives any serious problem to your future work.

I feel even you have not given any treatment after treating with RNAse it works (check the manufacture's instruction about the activity of RNAse).

Cheers!!!

Hey martin,

I have also faced this problem many times.

I would suggest to do RNase treatment before PCI-CI step, as the more times you would do ethanol precipitation, the DNA loss would definitely occur.

Besides this in your case, it is sure that your eppendorfs or any other solution (Sodium acetate or any other) is causing problem. Try to autoclave eppendorf and make new solutions. Although loss would be there, but it can be decreased at some extent.

I generally go with 1st step. Do RNase treatment before PCI-CI step.

Good luck.

DNase contamination is the obvious reason for your reduced yield. You can either increase the temp to 60oC when the Rnase will be still active but not the DNase or you can include a small concentration of EDTA which will scanvenge the divalent ions helping the DNase.

May I point the esteemed readership to a really old article I wrote way back then:

Heinze, B. (1994). RAPD reactions from crude plant DNA. Molecular biotechnology, 1(3), 307-310. Abstract: As opposed to standard polymerase chain reaction (PCR) using specific primers, genome analysis involving short random primers, for example RAPD, may yield inconsistent results if crude plant DNA preparations are used as the template. When RNase A, a thermostable enzyme, was added to such reactions, highly repeatable banding patterns were obtained from crude plant DNA, thus speeding up analyses substantially.

Sorry for the self-ad, but some of the findings there seem to still be relevant here ...

Hi all and thanks for the comments. I'll try to reply to some of them, and also summarize experiences that others have share on an email list.

Andrija, this issue is not confined to a particular batch of the Qiagen RNase A (the stock was freshly ordered), but has happened in different batches. This has also been confirmed now by several other users, and it seems like the Qiagen RNase A generally has this effect on already extracted DNA. I would therefore avoid using it. Whether this is because it isn't truly DNase-free or not, I am not sure, but I guess it may be the reason. Other users, which have also struggled with failed DNA samples after Qiagen RNase A treatment have later successfully treated their (already extracted) DNA with Thermo's DNase-free RNase A.

Thanks for the alternative suggestions to inhibit the activity of DNase (especially Siva; increased temperature and/or addition of EDTA during enzyme digestion) or lower the DNA loss by longer ethanol precipitation (Bastian)! Or degrading DNase by boiling the RNase A (Cynthe).

As stated in the original post, it is of course better to perform the RNase treatment on digested samples before the actual DNA extraction (Phenol-Chloroform in our case), but this is not an option for these already extracted samples for which no original blood sample is available.

Jetty, the DNA will be used for RAD sequencing, for which optimal yield of even representation between individuals among RAD tags high molecular weight DNA is preferred. I agree that for regular PCR's for Sanger sequencing this would not be an issue at all. Performing another Ph-Chl extraction of the samples after incubating the RNase A might be an option. Berthold and Jetty, the RAD sequencing is (as you may know) based on a first restriction enzyme digestion, so this publication probably has some bearing!

Smitta, it is high molecular weight DNA, so it definitely wouldn't denature at 37 degrees!

Cheers,

Martin

Hi Martin, spot on! You can always boil it (Anfinsen got Nobel Prize because of RNAse stability). This should denature and remove DNAse, but still some other endonucleases may be heat resistant. Personally, I never use RNAse in the last step of DNA extraction and it always needs to be removed in subsequent steps.

Cheers

Andrija

Anyway, though your DNA is partly degraded I hope it should certainly possesses some of RAD tags that you are going to flank (no idea what restriction enzymes are you going to use). Wish your RAD tagging should go smoothly.

Good Luck!!

From what I can see the only explaination is that some how you are contaminating your samples with dnase

Hi Martin,

Thanks for sharing your question, I am having exactly the same problem as you, and Qiagen RNAse is making my DNA from precious samples disappear. Just wondering what solution you came up with? Did you end up using a different brand of RNAse with success, or boil the RNA, or change the protocol... or something else? I am finding it very confusing as when we use the exact same RNAse during our extraction protocol it works fine. I'm off to try boiling some now...

Thanks,

Jessie

Hi Jessica,

Sorry for not replying promptly. I'll give you two replies: Firstly, eventually I realized that for the RAD sequencing it was not really necessary to treat the samples with RNase A. In the end, this is what we ended up doing - avoiding RNase treatment. As long as you quantify the samples accurately (with a DNA specific assay, as with a Qubit), it doesn't really matter that there is also some RNA present.

Secondly, if you actually do need to treat your samples, and if they have been extracted previously, there's no idea trying with Qiagen's RNase A. However, from other people that responded to a post on an email list, I concluded that they have successfully treated previously extracted samples with DNase-free RNase A from Roche and Thermo, respectively. I think that these protocols suggest an extra precipitation following the treatment, in order to get rid of remaining enzyme.

I hope that this information will be helpful, and I do hope it doesn't arrive too late...

Best,

Martin

Hi Martin,

Thanks for your reply. I ended up using a work-around also - I was attempting to get rid of RNA in order to pool multiple DNA extractions to send off for an illumina miseq run so I did need to get rid of the RNA - but ended up using a repli-g kit on one of the good (i.e. no RNA) samples instead... which worked beautifully...

Interestingly I did actually manage to get rid of RNA and keep the DNA using the Qiagen RNAse A by essentially re-extracting the DNA extractions that contained RNA using the Qiagen DNeasy kits, but I lost too much DNA along the way for it to be useful to me.

Best wishes,

Jessie

In Sometime, My F. oxysporum samples were isolated and extracted DNA from the samples with Qiagen RNAse, it making my DNA from previous samples disappears. 

Thank you for sharing this nice question and I wish you could make benefit from the remaining DNA samples after that supposed RNAse crackdown. Please, notice these reflections: 1) Have you been formally informed about the protocol of using RNAse A as explained by the manufacturer, i.e. is it good for treating DNA obtained by a Phenol-Chloroform protocol? 2) You said that your DNA samples gave large bands of high molecular weight DNA in gels; was that not satisfactory enough to start you downstream expreiments? 

Does any one know when RNA-free DNA is really needed in any downstream protocol?

Hi everyone for the past 3 weeks I've been battling with  PCR. The extraction is mostly a success, however, 16S PCR just seems to not work. I've changed primer concentration, Nuclease-free water and the concentration of my DNA template. Please help thank you :/ conditions being as programmed to perform 35 cycles of 95• (5 min); 95• (30 sec); 53• (30 sec); 72• (1 min) and final stage 72• (10 min). PCR RR mix PCR Master mix 12.5, Nucle-Free Water 8.5, RP 1, FP 1, and DNA template 2 microlliter = 25 PCR RR MIX

Dear Audrey, please post your question as a new question, not as a reply in a totally unrelated thread. Good luck with your PCRs!

Following up on this question: you may have diluted out your RNAse. OP said they used 1 ul of  10 μg/ml RNase A, but most of the protocols I've seen use 1-100 ug/ml FINAL [ ].

Dear Alejandro,

We did follow the manufacturer's instructions. As evident from the other replies, however, the original problem is neither uncommon nor unknown. DNase frequently co-purifies with RNase (even in products labeled DNase-free), but is much less thermostable. Thus, a simple solution is to boil the RNase for 10 minute before first use, let it cool, and remove any precipitate.

Article Unexpected DNA Loss Mediated by the DNA Binding Activity of ...

I had the same problem of loosing target DNA after RNase A treatment during plasmid miniprep in spite of following instruction of manufacture's directions. I would autoclave RNase A to remove DNase contamination, add 2 mM EDTA to the reaction mixture during RNase digestion

Hi Aly,

Yes, as has become evident in this thread, boiling the RNase A to kill off co-purified DNase should be standard (and really should be advised by the manufacturers!). How does the addition of EDTA to the RNase digestion improve performance?

EDTA is a chelating agent that removes the prothesic metal ions (zinc) from the DNase thus inhibits its action.

This thread did solve most of my query. In Summary I understand that Roche or a Thermo RNase A needed to be used ( Correct me if I am wrong). Also that the RNase A needs to be boiled and precipitate discarded before used. Is a protocol available for use of RNase A, if I have to use my sample to rid of RNA before the DNA library prep?

Vinayak Joshi: I have never had any problems using the RNase A during extraction (in connection to lysis step), only when applying it to previously extracted DNA. Based on the replies in this thread, and subsequent use, it seems like the problem definitely was that DNase had co-purified with the RNase (even though it was supposed to be pure). Luckily, the simple solution was to boil the RNase A. If precipitate forms, remove that, but for me that has never happened. As long as you boil the RNase A, I don't think it matters which brand you use. [Also, not all sorts of library prep really need RNase treatment, even if this is generally recommended in protocols and by sequencing facilities...] Good luck!

To remove DNase contamination from RNase treatment step, I would suggest heating up you DNA preparation at 75C for 20 min to inhibit possible presence of left over DNAase