In a previous article I wrote a short primer on soil molecular biology, introducing some of the issues surrounding isolation of DNA and RNA from soil samples, such as PCR inhibitors and lysis considerations. Today I will go into greater detail on the isolation of DNA from microbes in soil and how to optimize your results using the MO BIO PowerSoil^® DNA Isolation Kit.

Because of the amount of detail I want to share with you, this will be a two part blog. The first part will focus on the steps involved in homogenization, that being the beads, the homogenization equipment, and the lysis buffer. In the second part of the article, we will focus on the role of the chemistry in achieving optimal binding, washing, and eluting clean DNA.

MO BIO Laboratories has several products for DNA isolation from soil. For the purposes of this article, we will focus on the PowerSoil DNA Isolation Kit because it is our most popular product for this purpose. It uses our patented Inhibitor Removal Technology^® (IRT) for the removal of humic substances and polysaccharides and is performed using a mini spin filter and a microcentrifuge. 

Important notes before starting…

Something to keep in mind is that all soils vary in microbial load and organic content so DNA yield among different soils can vary. Yield is not based on the amount of material processed alone. Even soils collected from the same core but at different depths within the ground will have variable load and organic makeup.

For better consistency…

Consistency in yields between preps is difficult to achieve with soil because each scoop can contain different amounts of organic material, such as plant leaves or debris, insects, pebbles or sand. At MO BIO Laboratories, we sieve the soil for the best consistency so that the texture is uniform and the large particles are removed. If uniform yield among your preps is important to you, sieve first.

More is not always better….

It is important to note, processing more soil does not always yield more DNA. This is because the lysis buffer will be absorbed by the bead solution making sample homogenization inefficient.  Scale up of soil is possible but is soil-type dependant.  The PowerSoil DNA Kit is meant for small scale preps. If you need to process more soil than 0.25 grams, MO BIO offers alternative kits such as the PowerMax^® Soil DNA Kit for 10 grams of sample and the RNA PowerSoil^® Kit with DNA Elution Accessory Kit for starting with 2 grams of soil.

Now let’s go over the DNA Isolation protocol step by step. We will talk first about lysis and specifically about the mechanical aspects of lysis.

Step One: Lysis

High yields of high quality intact DNA requires a strong lysis. The lysis needs to be strong enough to break open microbes and fungus without severely shearing the DNA. There has to be a balance between the types of beads used and the amount of time used for mechanical homogenization. Temperature can also be used to boost lysis of tough organsisms or spores in combination with bead beating.

Bead types: 

There are many bead choices for the lysis of microbes in soil. The type of bead, shape, and size will all impact the DNA yield and integrity.

MO BIO prefers to use a garnet rock type of bead that varies in size and has sharp edges. Because of the size variation, the rocks can help break down both large clumps of soil and grind microorganisms that shake loose. Garnet is soft so will break down into smaller pieces when used in a high powered bead beating instrument. This works fine as many of our customers use them in the FastPrep or Precellys when they want to increase the lysis power for isolation of DNA from fungus (references below).

Other beads may be used, such as 0.5 mm glass or 0.1 mm glass if bead beating in a high powered instrument for longer periods of time is desired. These beads will cause more DNA damage but for very tough organisms, such as spores, it can be helpful. You can even mix the glass and garnet together if you need a combination of large and small

The homogenization equipment:

As described in the previous article, the vortex homogenization method allows for the best integrity DNA and is also the least expensive method. The time for vortex is ten minutes and this gives optimal results for lysis of bacterial cells in 0.25 grams of soil in our lysis buffer. Longer vortex times do not seem to increase the yield and will cause more DNA shearing.

The use of a Precellys or FastPrep is an option if you have one and want stronger lysis. Most customers use these for only 45 seconds to 1 minute for isolation of bacterial and fungal DNA at a setting of 5 on the FastPrep. A setting of 5 m/s on the FastPrep is equal to about 5200 rpm on the Precellys. Some customers prefer using a FastPrep setting of 4 (Precellys setting of 5000 rpm) for 15-30 second intervals and 3 or 4 pulses per sample. As you can see, using a high powered bead beater will require some evaluation on your part to determine the best setting for your sample. A list of references where the FastPrep was used in combination with the MO BIO UltraClean Soil or PowerSoil Kits is at the end of this article.

The lysis buffer: 

The other key ingredient in a strong lysis is the solution used to pop the cells. This buffer needs to fulfill several functions when it comes to soil. First it needs to disrupt cell membranes in combination with the mechanical homogenization. Second, it needs to be gentle enough to not denature the DNA, and third, it needs to work regardless of the pH of the soil. Soils that are acidic need to be neutralized for optimal DNA yields since the acidic conditions are harmful to the DNA. The lysis buffer in combination with the Solution C1 or S1 (in PowerSoil and UltraClean Soil kits, respectively) provides the optimal conditions for microorganism lysis from any soil type.

What about heating?

For those cases where a stronger lysis is desired, besides trying a high powered beating method and glass beads, it can be helpful to heat the sample before beating. An incubation of the soil in the lysis buffer at 65°C-70°C for 10-15 minutes will help to weaken the cell walls before homogenization. This treatment has been effective for spores and fungus.

Another method is to perform freeze/thaw cycles (3) with the soil, alternating between -20°C or -80°C and 37°C. This can enhance cell breakage as well, although might be less convenient than simply heating as described above.

Summary:

OK, we will end here because this is a lot of information. To summarize so far, the lysis step is the area where the most optimization is possible and depending on what you want to do with your DNA, you can go as easy or hard as you need. It is the combination of the beads + the equipment + the buffer that works together to provide you optimal yields and integrity of DNA. Really, this applies to any sample you are lysing whether it is animal tissues for RNA, bacterial cultures for DNA, or biofilm for RNA or DNA.

Fortunately, MO BIO labs R&D scientists are spending a lot of time working out these optimal conditions for a host of environmental samples, saving you time for your experiments.

But we can’t work with every sample type so we would love to hear from you and how you optimized the lysis of your sample type for the best result. Let us know what you do; bead type, time and homogenization method, and which MO BIO Kit you use to get optimal yields of DNA or RNA.

Next week we’ll continue our discussion on DNA isolation from soil and focus on the steps involved in the purification from the silica spin filters.

Thanks for reading!

References for using  high velocity bead beaters and the MO BIO Soil Kits:

Shifts in Microbial Community Composition and Physiological Profiles across a Gradient of Induced Soil DegradationGuilherme M. Chaer, Marcelo F. Fernandes, David D. Myrold, and Peter J. Bottomley Soil Sci. Soc. Am. J., Jun 2009; 73: 1327 – 1334.

Variations in Archaeal and Bacterial Diversity Associated with the Sulfate-Methane Transition Zone in Continental Margin Sediments (Santa Barbara Basin, California)Benjamin K. Harrison, Husen Zhang, Will Berelson, and Victoria J. OrphanAppl. Envir. Microbiol., Mar 2009; 75: 1487 – 1499.

Diversity of Basidiomycetes in Michigan Agricultural SoilMichael D. J. Lynch and R. Greg ThornAppl. Envir. Microbiol., Nov 2006; 72: 7050 – 7056.

Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal FlowSteven A. Wakelin, Matt J. Colloff, and Rai S. KookanaAppl. Envir. Microbiol., May 2008; 74: 2659 – 2668.

Changes in Bacterial and Archaeal Community Structure and Functional Diversity along a Geochemically Variable Soil ProfileColleen M. Hansel, Scott Fendorf, Phillip M. Jardine, and Christopher A. FrancisAppl. Envir. Microbiol., Mar 2008; 74: 1620 – 1633.

Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a NurseryM. Filion, R. C. Hamelin, L. Bernier, and M. St-ArnaudAppl. Envir. Microbiol., Jun 2004; 70: 3541 – 3551.http://aem.asm.org/cgi/reprint/70/6/3541

Mycobacterium aviumsubsp. paratuberculosis in the Catchment Area and Water of the River Taff in South Wales, United Kingdom, and Its Potential Relationship to Clustering of Crohn’s Disease Cases in the City of CardiffR. W. Pickup, G. Rhodes, S. Arnott, K. Sidi-Boumedine, T. J. Bull, A. Weightman, M. Hurley, and J. Hermon-TaylorAppl. Envir. Microbiol., Apr 2005; 71: 2130 – 2139.http://aem.asm.org/cgi/reprint/71/4/2130

Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Different LifestylesJohan Dicksved, Helen Flöistrup, Anna Bergström, Magnus Rosenquist, Göran Pershagen, Annika Scheynius, Stefan Roos, Johan S. Alm, Lars Engstrand, Charlotte Braun-Fahrländer, Erika von Mutius, and Janet K. Jansson Appl. Envir. Microbiol., Apr 2007; 73: 2284 – 2289.http://aem.asm.org/cgi/reprint/73/7/2284