Given the length of the details, this article will be divided into two parts. The first part focuses on grinding beads, grinding equipment and lysis buffer for grinding homogenization. The second part introduces the selection of chemical solutions to obtain the best DNA adsorption binding, cleaning and extraction effects.
There are several kits used by MO BIO Laboratories to extract soil DNA. The PowerSoil DNA Isolation Kit is the best and most popular one. Therefore, in the following article, we will mainly discuss it. The kit uses a patented inhibitor removal technique (IRT technology) to remove humic substances such as polysaccharide polyphenols, using small centrifugal filter columns and microcentrifuges.
It should be noted that the microbial content and organic matter content of different soils are very different, and the DNA yield is also different. The yield is not only determined by the initial amount of the sample, but also the microbial content and organic composition of the samples from different soil depths in the same soil.
It is difficult to ensure consistent yields for each sample, as each shovel may contain as many leaves, wrecks, insects, and even pebbles and sand. In the MO BIO lab, we usually screen the soil samples and remove the large pieces to ensure that each sample has the same texture. Screening is a must if the consistency of the sample has a large impact on your experiment.
This is important. Increasing the amount of sample starting does not always mean that more DNA is available. Because too much sample absorbs most of the lysis buffer, the grinding efficiency drops dramatically. Increasing the initial amount of sample is only feasible for individual types of soil. The PowerSoil DNA Kit is a small extraction kit. If you need to extract more than 0.25g of soil, MO BIO provides 10g of soil like the PowerMax® Soil DNA Kit, RNA PowerSoil® Kit and the matching DNA Elution Accessory Kit to extract 2g of soil at a time.
Next, we follow the DNA extraction process step by step. The first is cracking, especially mechanical cracking.
Step 1: Cracking:
Obtaining high yields of intact DNA requires strong lysis. Choosing the type of bead and the mechanical grinding time requires a balance that minimizes shear damage to the DNA while opening the microbial cells. The use of grinding beads to beat the crushing, if combined with temperature changes, can enhance the cracking effect, which is very helpful for the cracking of hard organic matter and spores.
Grinding bead type:
There are many types of grinding beads suitable for cracking soil microorganisms. The type, shape and size of the beads will affect the yield and integrity of the DNA.
MO BIO prefers to use garnet beads with different sizes and sharp edges. Because the size of the beads is different, it can break large chunks of soil and take care of small microbial cells. The garnet is soft and will crack into many small pieces when using a high-speed bead mill. Many customers can also achieve good results in using FastPrep o or Precellys in order to enhance the mechanical cleavage of fungal DNA.
High-speed and powerful bead mill equipment requires long-term impact grinding, which requires the use of 0.5mm or 0.1mm glass beads. Although such beads can exacerbate DNA damage, it is helpful for the lysis of spore-like hard organics. You can also mix glass and garnets of different sizes to make a mix of beads for your own sample.
Grinding equipment:
As mentioned in the previous article, vortex grinding is the most cost-effective method of obtaining complete DNA. Experiments have shown that with 0.25 g of soil sample treated with our lysis buffer, a 10 min vortex time is most suitable. Extending the vortex time does not increase the yield, but rather increases the DNA damage.
Precellys or FastPrep is also a good choice if your lab has it and wants to enhance the cracking effect. When most customers extract bacterial and fungal DNA, they set FastPrep to 5 and grind for 45s to 1min. The 5th file on FastPrep is equivalent to 5200rpm of Precellys. Some customers prefer to use 4 files (equivalent to 5000 rpm for Precellys), 15-30s per sample, 3~4 cycles. It can be seen that with the powerful bead mill grinding equipment, it is also necessary to adjust the grinding scheme according to the specific conditions of the sample on hand. The literature at the end of the article lists some of the literature on using MO BIO UltraClean Soil or PowerSoil Kits on FastPrep for reference.
Lysis buffer:
Another important component that makes up the cleavage force to break apart cells is the "solution." This solution must meet 3 conditions. First, combined mechanical forces disrupt the cell membrane. Second, the soft nature does not damage DNA. Third, not affected by the pH of the soil itself. The acidity of the soil itself must be buffered because acidic conditions are harmful to DNA. Lysis buffer combined with C1 or S1 (PowerSoil and UltraClean Soil kits, respectively) provides the best conditions for lysing all types of soil microorganisms.
heating?
In the case where strong cracking is required, the high-speed high-power bead mill grinder and the glass bead are not mentioned, and heating the sample before grinding is helpful for the extraction. After access to the lysis buffer, incubation at 65 ° C -70 ° C for 10-15 min can weaken the cell membrane, which is good for grinding and crushing. It is especially effective against fungi and spores.
Another method is to freeze and thaw the soil sample three times from -20 ° C or -80 ° C to 37 ° C. This method also enhances cell disruption. Of course, it is not convenient to implement the heating method mentioned above.
to sum up:
Ok, I have already said a lot. In general, the lysis step is a combination of grinding beads + grinding equipment + buffer instrument to lyse the sample and greatly affect the yield and integrity of the DNA. Every aspect is very flexible, you can adjust according to the actual situation of the sample. Regardless of the extraction of animal tissue RNA, microbial pure culture DNA, biofilm DNA or RNA, the truth is the same.
Fortunately, MO BIO labs R & D scientists spend a lot of time optimizing the extraction conditions for various environmental samples, which in turn saves you valuable experimental time.
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