Which grinding method is best for different soil types

Author: Suzanne kennedy Source: US MoBio Laboratory [Font Size: medium and small]

We need to collect a large variety of samples during the product development process and then find the best microbial DNA extraction method. So when we develop PowerLyer's operational processes, we hope to find a way to fit a variety of samples. When we studied homogeneous grinding and grinding of bead casings, we knew that choosing the right beads according to the type of soil can increase the yield of DNA. So we decided to use PowerLyzer to do a similar study to answer a question: Is the same process flow on a strong bead mill using the same DNA yield and integrity results for different types of soil? Many people look for references to find the same soil type extraction method flow, and then copy it without optimization. However, can an extraction method be applied to all types of soil?

In the previous article "DNA Extraction I: Molecular Biological Properties of Soils", we have a comprehensive understanding of the soil homogenization. Today, I use examples to answer these questions. We used a high-speed grinder (PowerLyzer) to compare the DNA yield and integrity of two different types of soil, one with high clay content and one with high organic carbon content; two different grinding beads were added: 0.1mm glass beads ( Item No. 13118) and 0.7mm garnet grinding beads (Cat. No. 13123). The result is amazing.

Research methods:

We received different types of soil samples from the Soil Research Center of the California State University and selected two soils with similar clay content and different organic carbon content. One of them contains 45% clay and 2.5% organic carbon; the other contains 40% clay and organic carbon up to 5%. The higher the organic carbon content, the more biomass, which means the higher the final DNA yield.

For comparative experiments, a 0.7 mm garnet bead from the PowerSoil DNA Isolation Kit and a 0.1 mm glass bead from the PowerLyzer PowerSoil DNA Isolation kit were selected. A series of grinding tests were performed on the PowerLyzer from 2000 RPM to a maximum speed of 5000 RPM. Each sample was ground for 45 s.

The DNA extracted from the PowerSoil kit was measured with a Nanodrop spectrophotometer and run for electrophoresis to check the integrity. The results of the glass beads were marked in blue, and the results of the garnet beads were marked in red to make the bar chart below.

result:

Clay, low organic carbon content soil:

From the first figure, the high clay content soil yield is higher. We can see that the glass beads can achieve the highest DNA yield (bands 14, 16) at 3900-4200 RPM. Interestingly, when the grinding speed exceeds 4200 RPM, the yield does not rise and fall. It has been proven that over-grinding soil samples can lead to DNA loss. That is to say, to obtain the highest yield of DNA, there is a grinding speed and an upper limit of time.

We can also see that at less than 3900 RPM, both the ground bead and the garnet extract have similar DNA yields. Why is this happening?

There is a theory that the two main components of the soil are easily cracked and difficult to be cracked. At low speeds, both beads are easily smeared with easy-to-crack components, while at 3900 rpm the glass beads lyse another subset. This part may be fungi and spores.

However, when the grinding is too much, the DNA obtained in the front is severely degraded and degraded, resulting in a low yield.

Loose soil with high organic carbon content

The second picture is the same experiment done with a similar soil with a high organic carbon content. The clay composition is as high as 40%, although there is no high content of 45% of the clay in the first sample, but different results are obtained.

Garnet beads and glass beads were similar in DNA yield at the beginning. But when it reaches 3200-3500RPM, the performance of garnet is better than that of glass beads. However, in the electrophoresis, it is seen that the DNA damage is relatively serious. A fragmented DNA fragment pushes up the OD value and is mistaken for an increase in yield. Therefore, we have always recommended that it is best to measure OD and combine it with running electrophoresis to check DNA.

There are many reasons why the OD value does not fully reflect the true DNA yield, see Popal Misconceptions about DNA Isolation and Quantification.

For this type of soil, the overall DNA yield is still high. The use of garnet beads yields the desired DNA yield at a range of speeds and peaks at 4200 RPM, with subsequent yields not increasing with speed. These data prove that speed and time have reached the limit and no more DNA can be extracted.

to sum up:

1- The above data tells us that soil types are diverse and each has its own unique characteristics. The extraction of sand samples from beaches is certainly not the same as that of forest soils, and there may be differences between each clay sample. The consistency of the texture of the soil sample, the difference in biological abundance, and the inconsistent organic content will affect how much DNA and the choice of grinding method you can ultimately obtain.

2- Run electrophoresis combined with OD measurement is the most important and most commonly used method for soil DNA evaluation. Soil samples were extracted using the PowerSoil DNA Isolation Kit and only DNA was extracted without any RNA. Other methods on the market, along with RNA, extract total nucleic acids, greatly pushing up the OD value, and mistakenly believe that the extraction process is successful. It is possible to detect the integrity of DNA by running electrophoresis, so that it is a bit too much to know that the grinding power is not enough.

3- Because each soil type is different, it is necessary to perform a preliminary experiment through a series of different speeds, grinding times, and even different types of beads. The PowerSoil Kit is recommended starting at 4000 RPM 45s because this speed and time is the most suitable for two different beads and most soil type samples. But maybe you want to turn the speed down or up, depending on the spore content and microbial abundance in the sample. Try a series of experiments at different speeds to know at which speed your sample gets the most high quality DNA.

Chris Kitts lab of California State University participated in the study with us. The students completed t-RFLP analysis of DNA extracted from five different types of soil using Vortex and PowerLyzer, garnet beads and glass beads. Click to view related posters.

Finally, it is recommended to use your grinder for a linear speed study before starting a new project. See if the previous experimental solution for soil use is appropriate and whether new samples need to be further optimized. Different soil characteristics are unique, and it is worthwhile to spend more time in the early stage to secure valuable data.

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