I am trying to extract DNA from a variety of geothermal soils for metagenomics, but I am struggling with low pH (pH4, so I can get rid of some of the humic acids and other inhibitors with these methods, but not at low pH.
I've heard the Mo Bio Power Soil kit is good with clay - does anyone know if it works well with low pH samples?
I've used the PowerSoil kit to extract DNA from samples with even lower pH (pH ~2.0), but not clay soils or similar.
However, your problem might be related to the adsorptive capacity of the clay more than anything else. I recently found a paper (see link below) that uses a clay desorption procedure on cemented clays improving the output. I haven't tested it but it may worth take a look.
Article Improved Yield of High Molecular Weight DNA Coincides with I...
Hi Karen
Give the above mentioned article a try. We took a couple of years to develop it and it does work on the clays. It is an involved procedure no doubt but the key is the 1M bicarb to prevent the DNA from the lysed cells from sticking to the humic acids. Once they stick, they are essentially gone at least in terms of getting the non-fragmented DNA back for analysis.
Power Soil may not be the best protocol for soil with low pH (see article from Sagova-Mareckova et al 2008).
For soil with high clay content, you can also have look on the article from Hale and Crowley (2015) who developed a protocol for soil with amended-biochar sand or clay.
Finally, have look on the article from Terrat et al (2012) comparing protocols for different soil (including clay) for metagenomics.
And as previously mentioned, the article from Hurt et al 2014 is a good starting point.
Have you tried diluting the DNA templates before PCR? It helps eliminate inhibition.
Thanks for the replies so far. The problem seems to be extracttion rather than inhibition - I've tried loading the entire "DNA" extracted onto an agarose gel but there's no DNA there - I think it probably degrades as soon as the cells lyse.
I've tried adding bicarb to the cells before lysis but this didn't help. Does anyone have any other suggestions for buffering the cells before lysis to keep the DNA intact?
Try the new PureLink Microbiome DNA Purification Kit – http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product
Dear Karen, it has been 3 years. Did you find any working solution in the end? Thank you in advance for sharing!
ok, this is our solution for the problem after trying all the suggestions above, and many other things. Try a newly developed Soil PRO (!, not PowerMax, not PowerSoil but "PRO") kit by Qiagen. Good yield and purity for DNA isolated from clay up to 120 cm deep.
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