I don't have any experience of soil DNA, but I had to make some extractions. I used ZR Soil Microbe DNA MiniPrep kit from Zymo Research. For the first sight, almost all samples were OK, only some of them appeared degraded. I made an electrophoresis after some months of storage, and I realized that most of my samples were empty. I tried to figure out some tendency, for example degradation of samples from the same soil types, but actually I could not conclude anything. Does anyone have any suggestion on this problem? Is it a common phenomenon? Is there enzymatic DNase activity that remain in the sample, or is it rather a chemical degradation?
Hey,
in fact, also DNA can be degraded over time, but it has been shown that it can also absorb on different soil mineral particles and thus be not available anymore. What solvents did you use for extractions and how did you store the soil prior to your measurements? Was the soil air-dried or wet?
Attached you find an example for the mentioned processes.
http://aem.asm.org/content/67/1/293.full
Thank you for your answer!
With DNA from plant source I have never faced this degradation problem, years after the extraction the DNA was stable, visible on gel and could be used for PCR or so. Ok, I don't know, if my soil samples were usable for downstream applications, but at least, they were visible on gel.
The soil I used was wet. I used reagents from that above mentioned kit, and the producer tells nothing about their composition. This was a column-based kit. I made the elution in water, and did gel-electrophoresis right after the extraction first. Then I stored the samples on -20 degrees, for two month, and made a second electrophoresis.
Dear Rita,
My ex-boss used to store DNS samples at +4 degrees for tens of years (even 30) in TE. Usually when using comercial kits I use elution buffer for DNA only in case it is TE. EDTA prevent DNA activity and do not inhibit PCR in that concentration (even at 10x concentration).
I recommend, simply use TE for elution. pH is important to be around 8-8,5.
Soil work I do not know. If your problem comes from soil characteristics. others will hopefully tell some usefull hints.
Best,
Feri
I was also wondering because normaly it takes really a long time, even in soils, until DNA is degraded. You should at least see fragments of your original molecules. But i have no experience if sorption processes in soil constitutens can so strongly influence the measurability, i would rather expect a minor role.
Feri, thank you for your suggestions! We preferred to avoid EDTA to date . Of course, it might be a superstitious practice :)
Christian, I am really confused, I have quite a lot of experience with DNA work and I saw DNA degradation only in dried plant samples. I suspected that probably humic acid or other chemical contamination released from the soil lead to the damage of the DNA, but unfortunately I have no experience with soil-work. The other hypothesis was that DNAse activity remained in the sample (and no EDTA added to restrict it).
I have several extraction from the same plantations, and I wanted to pool the samples and concentrate DNA. But now, I am afraid that I might cross-contaminate my "good" samples and loose everything.
Attached you find a good paper dealing with the stability of DNA in soil and occurring processes! Maybe it helps you identifying the problems with your samples. You wrote that the soil was not completely dry during the storage?! I'm not sure but maybe some reactions could still have taken place (biotic / abiotic), although it was stored at -20° C (which is rather unlikely).
You could also ask someone which is really familiar with DNS extractions from soil matrices, e.g. Doreen Babin from the Institute for Epidemiology and Pathogen
Diagnostics at Julius Kühn Institute or Michael Hemkemeyer from Thünen Institute of Biodiversity.
Good Luck!!
Thank you Christian! In fact, I stored soil samples on 4 degrees and stored soil-DNA samples on -20. So the stability problem occured in the extracetd DNA, they degraded during storage.
Thank you for the paper, it looks quite helpful!
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