I am extracting the soil DNA from fertilized and biochar amended soil. The samples were taken one year ago and stored at 4 degree celsius. Now I am going to extract the soil DNA using MOBIO Power Soil DNA ext kit but the quantity is very low (it ranges between 3-8 ng/micL). Where is the error and how can I isolate large quantity of DNA?
Thanks
Probably, the biggest mistake was to store the samples at 4 degrees for one year.
It would be better if you stored the samples at -20 or (much better) at -80, the same day of sampling.
Furthermore, it would be better if you freeze-dry your samples.
I don't know if you followed exactly the protocol from MOBIO, but you could obtain a better result if you use 0.25 g of soils, instead of 0.50 g: the buffer absorption in the beads tube will be less and you will have a more efficient cell lysis.
In my experience it worked fine.
Thanks Andrea, Finally i extract the DNA using 0.5 g soil Sample. Did you think, storage of samples at 4 degrees will effect the changes in microbial communities? what changes can occur?
Hi Muhammad, at 4 degrees the cell metabolism is slow but not absent. Furthermore, probably you stored the soil in closed bag, so you created an anaerobic environment.
You probably artificially selected microorganisms that are able to survive in anaerobic conditions and at low temperatures, losing in the meanwhile the strict aerobics and the mesophiles.
For sure the microbial community is altered, lot of papers recommend to freeze-dry the soils samples in the same sampling day, if possible.
However, you can still try to use liquid nitrogen to freeze the samples, grinding the material with mortar and pestle (100-300 micrometer sterile glass beads are very useful to obtain good results) and then you can try to use less soil (0.1-0.25 g).
I'm quite sure you will obtain better results!
Dear Muhammad Azeem,
I am agree with the idea of the Hani Antoun, it would be very tedious task, to analyse the microbial communities in the soil sample, you have stored at 4 degree C. However if you want to try then you can try it with lesser amount of soil sample with at least 30 minute vortexing with the beads in the lysis step of the protocol.
I am afraid I have to agree with Hani and Andrea - you will get DNA (and ultimately data), but that will be the DNA of microbes from those soils that liked the conditions you inadvertently incubated them in for a year. By all means use these samples as a trial set for you to practice on, but you won't (and quite rightly, IMO) be able to publish the results - the data will be of no value.
I appreciate that this probably isn't the answer you wanted, but use this experience to learn, and next time either freeze the samples (-20 should be OK for DNA, but flash in LN2 and -80 if for RNA), or extract them immediately. The community is very dynamic, so you need to ensure that you're measuring the microbes that were there when the sample was in situ, not the microbes that are there after storage.
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