The DNA needs to be pure, clean, free of inhibitors and suitable for quantitative PCR. The current method we use is limited to 0.25g of soil and I would like to use at least 10g, more if possible.
This may be helpful (available on RG):
Haling, R.E., Simpson, R.J., McKay, A.C., Hartley, D., Lambers, H., Ophel-Feller, K., Wiebkin, S., Herdina, H., Richardson, A.E. & Riley, I.T. 2011. Direct measurement of roots in soil for single and mixed species using quantitative DNA-based method. Plant Soil 348: 123-137.
I hope this maybe useful: Plassart P, Terrat S, Thomson B, Griffiths R, Dequiedt S, Lelievre M, Regnier
T, Nowak V, Bailey M, Lemanceau P, Bispo A, Chabbi A, Maron PA, Mougel C, Ranjard
L. Evaluation of the ISO standard 11063 DNA extraction procedure for assessing
soil microbial abundance and community structure. PLoS One. 2012;7(9)
In theory, any protocol could be used for larger amount of soil than 0.25g; you “just” need to upscale everything (tubes, purification columns, volume of solutions…). There are few DNA extraction kits available on the market for amount of soil >0.25g, but nothing above 10g (that I heard of):
- PowerMax Soil DNA isolation kit (up to 10g of soil) from Mobio
- EZNA Soil DNA Kit (up to 1g) from Omega
- NucleoSpin Soil (up to 0.5g) from Macherey-Nagel
http://www.mobio.com/soil-dna-isolation/powermax-soil-dna-isolation-kit.html
http://omegabiotek.com/store/product/soil-dna-kit/
http://www.mn-net.com/tabid/11352/default.aspx
The first thing to do to dry the soil, may be overnight at 40degree C, use plastic beads (0.6mm) and SDS based soil extraction buffer. After bead beating and spinning, take the supernantent and add ammonium acetate (10M), incubate in fridge 1/2 to 1.00 hour, spin and transfer the supernatentto new tubes and discard the pellet.
Extract DNA from the supernatent by usin PCR cleaning kit.
We have been practicing this method in our lab on a routine basis and the extracted DNA is used in qPCR and standard PCR. The protocol can be used successfully from a small soil sample to a large (.05g-100g) with modification according to the size of the sample.
Upscale the protocol used in this paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC91286/ you may combine it at the end with a PVPP column step to get it absolutely contamination free http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1996.tb08128.x/abstract
Thanks to everyone for the information, this is really helpful.
Farhat- can you give me a bit more detail about your protocol? What exactly is in the SDS buffer, how much to add, how much ammonium acetate to add, how long to spin for etc.
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