Dear Evoldir Community, We've had some very interesting responses to our problem regarding our disappearing DNA. Below, please find all responses on the matter and more. Thanks to everyone for all their help, Nicole Foley *Original Query* Dear Evoldir Community, Recently in our lab we have been experiencing intermittent problems with long term DNA storage. The samples causing problems would have been extracted using a variety of different methods, stored at 4 degrees during use and then moved to -20 or -80 degrees for long term storage. These samples all have good nanodrop values and have been successfully PCR'ed. Once the samples are taken out of long term storage and are nanodropped again the DNA seems to have disappeared!!! The samples have been stored in a variety of plastic ware (different types of tubes and plates) all made from polypropylen. While it is possible that the DNA is somehow becoming stuck to the plastic, the fact that the problem is happening so infrequently makes us think it may be something else. Thankfully we have managed to recover the samples by heating and shaking at 60 degrees continuously for ~48 hours, but are puzzled as to why this is happening in the first place. Has anyone else experienced this before or have any insights into the causes? Thanks in advance for your help and I will of course re-post responses, Nicole Foley *Responses* It could simply be that the samples stored below -20C become insoluble and the DNA tends to crystallize to the walls of the tube. Thus you cannot pick up any signal on the nanodrop and when you re-suspend the DNA you find it again. Another cause could be the quality of the buffer you are using and the tubes you store the DNA in. You could either precipitate the samples and store them dry or just keep them at 4C - DNA is generally quite stable. Peter shum.p@hotmail.com Hi Nicole I have seen good DNA go bad but not since I started using low bind plastic. I use eppendorf lo-bind tubes and twin.tec plates. The plates are designed for PCR, but also work fine for DNA template storage. But I know from others that it is not always the solution. Sometimes DNA just mysteriously disappears. http://www.eppendorf.com/int/index.php?sitemap=3D2.1&action=3Dproducts&contentid=1&catalognode=14136 There is an interesting paper by Gaillard and Strauss about this issue: international biotechnology laboratory / august 2000 Eliminating DNA loss & denaturation during storage in plastic microtubes good luck Arjen A.E.Van-T-Hof@liverpool.ac.uk My lab has experienced this from time to time with a small number of samples. In all cases, the DNA had been stored in water rather than a buffer like TE. DNA eventually denatures in water, especially if dilute. It can often be (partially) renatured by treatments similar to the one you used, and this can be facilitated by adding a small amount of concentrated TE (or that old standby, SSC) to the denatured DNA sample before heating. It is less successful if the storage water was acidic (below pH of about 6.0) to begin with or has become so with time or upon initial thawing from the freezer. Our Sanger sequencing facility on campus (like many others) requires samples to be submitted in water. There is a temptation therefore to elute our fish DNA samples from purification columns with water and then store them that way. If such samples are stored at - 20 or even -80 C for some time, we encounter the denaturation problem. So with precious samples we elute withTE (or with the elution soln. provided by the supplier of the columns, generally Qiagen or Zymo) and then repurify an aliquot and elute with water for sequencing. Bruce Turner fishgen@vt.edu Dear Nicole Foley, I would like to share with you my experience: nanodrop measurements are not very reliable. Sometimes at all. I don´t know how old are your DNAs, however, my DNAs were PCRable after 4 years of -20 storage (very good amplification at variable loci). Another option might be to lyophilisate DNA for long-term storage and keep it at -20 or - 80 °C. Then dilute when you will need this DNA again. Just an idea. Nothing really new from my site, perhaps others will have bigger expertise in DNA storage. All the best, Josef Janoušek Mendel University in Brno, Czech Republic janousek.jose@gmail.com Hi Nicole, How was the DNA extracted and what is it stored in (e.g. TE, water, etc)? Some extraction methods such as boiling are good for short term yield, but can damage the DNA. Similarly some may leave other compounds in the solution that degrade the DNA. The final solution it is stored in is also important as it needs some buffering capacity. Theresa theresa.burg@uleth.ca Dear Nicole That's not a big problem at all, we know DNA is a macro molecule and has a density more than water. Since we are storing these DNA micro centrifuge tubes (or any other) for years in a vertical position all the DNA may have settled down. You might have thawed and checked the nano drop reading again, but the quantity we generally load on nanodrop is to less and there is a possibility that it has not become an uniform DNA solution with simple thawing or vortexing. It's good to hear that you managed to recover them by mixing and heating which also suggest us that the DNA has gone no where....!!! Keep posting these interesting observations!!! Cheers Pranay pranay.amrut@gmail.com Hi Nicole, Thanks for posting about your problem on the Evoldir weblist. I work with conifer DNA and this problem happens to me too. My DNA is stored at -20 degC all the time. When I extracted it it was in high concentration but a few month later there was only a little left in all of the tubes... I thought it was due to contaminants degrading the DNA in some way (conifer cells are full of crap!) ... But the fact that it happened to you (What organism are you working with?) and especially the fact that you recovered it by heating and shaking makes me reconsider my interpretation! Also it might mean that it is a more common problem than I previously thought... Did you get replies other than mine? Also, If you need to continue investigating these kind of issues in the future, I would suggest you use the Qubit concentration reading method, more accurate in reading DNA or RNA concentration... it is more expensive than the nanodrop though, as it uses special reagents. Thank you, good luck, and all the best for your research! Joane joane.elleouet@gmail.com I hope you'll post your answers I've actually had the strange result that DNA stored in the fridge fared better than DNA in the -80. I had purified plasmid DNA in water, stored for about 4 years - i was able to sequence the plasmids stored in the fridge, but those stored in the -80 failed. I know that frequent freezing and thawing of genomic DNA tends to shear the DNA, so I keep any working solutions in the fridge, but these samples were only frozen once. Of course it could be some other difference in the template that cause the frozen ones to fail.. best wishes diana dewolf@alaska.edu Hello Nicole, I am TA in the Meyerlab. I heard that you have problems with your DNA. I found a video from the Company Biomatrica maybe it could be a solution for you. https://www.youtube.com/watch?v=3DyJTcHgEZaKg You can ask this company why you have this problem with the DNA. here an link from the Company Qiagen: https://www.qiagen.com/de/resources/molecular-biology-methods/dna/#Sizes%20and%20molecular%20weights%20of%20various%20genomic%20DNAs Have a nice day, Dominique dominique.leo@uni-konstanz.de Nicole, We tend to only store RNA at -80, not DNA, because we have found that temperature to cause breaks in DNA. -20 seems fine for long-term storage, especially if your DNA is in TE or another buffer. I haven't done anything rigorous to back this up, but that's been our experience. Chris clane@mail.uri.edu Hi Nicole, Polypropylene plastic ware can certainly cause issues for long-term DNA storage, due to DNA adhering to the plastic surface. A commonly-used way to both prevent this and 'rescue' extracts for which this has already occurred, is to add a detergent, such as Tween-20, to the extract after elution for a final concentration of 0.05% Tween-20. Hope that helps! Cheers, Pete Heintzman peteheintzman@gmail.com nicole.ma.foley@gmail.com