Subscribe to ArticlesToday we’ll pick up where we left off with our discussion on how to maximize the yields of DNA from soil. We have now thoroughly covered the subject of sample homogenization and the importance of the bead type, equipment, and lysis buffer. You can see that the lysis step is the most involved and the best place for optimizing yields.
Next, the sample needs to be cleaned before final purification. This is done using the Inhibitor Removal Technology (IRT) process. Continuing our article at the second stage of the protocol, inhibitor removal, let’s go into greater detail on the different aspects of the chemistry used for the purification of DNA from soil using silica spin filters.
Removal of Inhibitors:
We are now ready to remove the PCR inhibiting substances from the soil homogenate. The humic acids are what give the sample the brown color. Present will also be polysaccharide if your soil sample had plant material or even some biolfilm content. Removal of inhibitors is what makes MO BIO and the PowerSoil kits stand out. MO BIO Labs developed a patented method, called inhibitor removal technology (IRT) to precipitate out the humics, phenolics, and polysaccharides from lysates. IRT involves a two-step process where-by the proteins and debris are removed first followed by flocculation of large insoluble macromolecules. After using inhibitor removal solution (IRS), samples typically look clear. The PowerSoil and PowerWater protocols are all optimized for the amount of IRS needed to clear even the most problematic samples, however, more can be used if inhibitors are still present (as determined by PCR). Repeat flocculations with IRS are ok to do as necessary.
The IRT steps are performed using cold temperatures to enhance the flocculation but for the second step (IRS), we recommend not to extend the time significantly over five minutes. Lower DNA yields may result from prolonged incubation in IRS.
Stopping points?
If you need a stopping point in the PowerSoil protocol, the best place to pause is after the IRS step and before adding the binding solution. The lysate can be frozen at -20°C and used for binding to silica spin filters the next day.
Binding to Silica Filter Membranes:
At this point, the DNA is ready for purification on a silica membrane. The lysate should look clear (can be slightly yellowish if the soil was heavy in organics). For the DNA to be captured on silica membranes, it requires the presence of a high level of chaotropic salts. The ratio of the binding solution (Solution C4) to the lysate is critical for good yields. If too much is used, recovery of degraded RNA will result. If too little is used, a portion of the high molecular weight genomic DNA is lost. For this reason, we instruct you to take up to 750 µl of your lysate into this step so that the entire lysate will fit in the 2 ml collection tube once the 1.2 ml of binding salts are added.
If you need to take more than 750 µl, you will need to increase the binding solution as well. A good ratio is two volumes of binding solution C4 per sample volume. You will need to split the sample into two 2 ml collection tubes or a larger tube (5 ml or 15 ml) to make sure everything is well mixed.
Vacuum Manifold Option:
Normally, if you followed the standard protocol, binding to the spin filter requires three loadings of the column. One way to speed this process up is to try the PowerVac Manifold System. It is very fast and easy and results in less handling. If you have a vacuum manifold already, then all you need are the PowerVac Mini Spin Filter Adapters. In our lab, we regularly use this method to speed up processing. If you decided to use more of the lysate than recommended and increased the amount of binding salts, using the vacuum manifold will be the best way to reduce the time required for loading the column 4 or 5 times.
Washing the DNA:
Because of IRT, most of the soil related contaminants are removed so the column will not need a heavy salt wash like with other kits. The washing step here is needed to remove the chaotropic salts from the column. If any salt is left behind on the column membrane, the DNA will not elute efficiently and the DNA that does elute will be contaminated with guanidine. To remove salts from the column, the wash buffer contains ethanol which solubilizes and rinses away salt. One wash typically does the trick. However, if you are having problems with low 260/230 readings (as observed by high 230 absorbance on a Nanodrop), then a second wash may be performed. If you run out of wash buffer, 100% ethanol can also be used to wash the membrane as well. We use 100% ethanol on the vacuum manifold protocol and this can be used manually in the event you run out of wash solution and need more.
Don’t forget to spin dry the column before elution so the DNA can be eluted efficiently. Left over ethanol on the column will make the DNA release from the membrane inefficient.
Elution:
The final step is releasing your DNA from the membrane into a 10 mM Tris pH 8.0 buffer. DNA dissolves faster in a neutral to slightly basic pH. You may use water to elute but because water tends to have a low pH (usually around 4-5), the efficiency could be reduced. One hint for an increased yield during elution is to allow the buffer to incubate on the membrane a few minutes at room temperature before centrifugation. Incubation from 1-5 minutes will help resolubilize the DNA in a smaller volume. Don’t elute in less than 50 µl or you will leave too much DNA behind.
Your DNA is now ready to use in PCR or for gel electrophoresis!
FAQs:
How much DNA is typically in soil?
After all of this discussion, you may be wondering how much DNA can I expect from soil? The answer is that it varies. The moisture content, organic content, and where collected will all play a role.
In our labs using “normal” soils or temperate soils, such as garden soil, the yields can range from 2-5 ug of DNA per 0.25 gram (a prep). We have worked with some agricultural soils, such as soil from the Strawberry Fields in Carlsbad, and these yields are far lower- around 0.25 ug per 0.25 gram of soil. Sandy and clay soils tend to have lower yields and very low organic content.
What can I do to increase yields in clay and sandy soils?
One current theory with sandy soils and clay soils is that the released nucleic acids are tightly binding to the soil itself. There are several references looking at ways to pre-block soils to prevent loss of the microbial DNA, including the use of skim milk (1). Some evidence suggests that divalent cations are playing a role in DNA binding to the surface of soils (2). For this reason, some of our customers have found success by adding EDTA into the bead tube during the homogenization step at a final concentration of 50 mM.
Summary:
This covers all of our tips and tricks for isolating DNA from soil. To summarize, soils vary widely in their characteristics and microbial load so expect the yields to vary when extracting different samples. Two key steps for obtaining high yields and integroty of DNA are the homogenization step and the binding step . If your yields are lower than expected, optimization is usually done at these steps. And remember, using more soil will not result in more DNA.
We are always interested in hearing how you tweaked the MO BIO kits for better results with your samples. Drop us a comment or email (technical@mobio.com) and let us know your special tips and tricks for isolating DNA from soil.
Thanks for visiting!
References:
1. Microbes and Environments
Vol. 19 (2004) , No. 1 pp.13-19
An Improved DNA Extraction Method Using Skim Milk from Soils That Strongly Adsorb DNA
Yuko Takada-Hoshino and Naoyuki Matsumoto
2. FEMS Microbiology Letters
Volume 97 Issue 1-2, Pages 31 - 39
Adsorption of DNA on clay minerals: protection against DNaseI and influence on gene transfer
Eric Paget, Lucile Jocteur Monrozier, and Pascal Simonet