Most of the time choosing a DNA Extraction kit is pretty straight-forward. If you want to isolate DNA from lung tissue, then use a tissue DNA isolation kit. If you want DNA from microbial culture, then use a microbial DNA isolation kit. But what if you want microbial DNA from the lung tissue? Do you choose the tissue kit or the microbial kit? And what if you want animal tissue DNA from a soil sample? Should you use a tissue or a soil DNA isolation kit? It gets a little tricky. These kinds of mixed up situations make up a good fraction of our technical support questions. We have worked out protocols for many of these scenarios. Below we discuss three of the most common mixed-up circumstances and give suggestions as to which kit and protocol will work best for them.
Microbial DNA from tissue
Kit to use: UltraClean Tissue & Cells DNA Isolation Kit
While traditional tissue kits are great for isolating DNA from eukaryotic cells, they usually aren’t so good for microbial cells. Microbial cells are harder to lyse. So customers are usually pretty surprised when we suggest they use our UltraClean Tissue & Cells DNA Isolation kit for bacterial or fungal DNA from infected animal tissue. A unique feature of the MO BIO UltraClean Tissue & Cells DNA Isolation Kit is the use of bead beating to homogenize the tissue. The bead tubes contain 0.7 mm crushed garnet bead tubes which are well suited for lysing bacterial and fungal cells. Both the animal and microbial DNA will be isolated with the kit. But with microbial specific primers one can usually get successful PCR amplification of the bacterial or fungal DNA of interest.
Tissue DNA from soil and feces
Kit to use: PowerSoil or PowerFecal DNA Isolation kit with change in protocol
What if one is interested in the DNA from organisms inside of soil like worms or maybe the DNA from human epithelial cells in feces? For DNA from Eukaryotic cells the best choice is normally a tissue kit. However, soil and feces are chocked full of nasty PCR inhibitors. A tissue kit just doesn’t have the chemistry to remove these. In order to remove inhibitors like humic acids and polysaccharides, we recommend a kit containing IRT, our Inhibitor Removal Technology. MO BIO PowerSoil and PowerFecal DNA Isolation kits contain IRT. But this presents another problem. The PowerSoil and PowerFecal kits were optimized for microbial DNA. They use bead beating and detergent for lysing bacteria and fungus. They do not use Proteinase K which is helpful to remove the genomic DNA from histones in the cell nucleus. (not an issue for prokaryotes which have no cell nucleus)
The way around this is to add a Proteinase K digest to the protocol. We suggest adding 20 ul of Proteinase K (20 mg/ul) to the bead tube at the beginning of the protocol and adding an incubation at 65 C for 30 minutes immediately after bead beating. One can lessen or forego the bead beating all together if there are no actual chunks of tissue to break down.
Bacteria and/or Fungal DNA from Plants
Kit to use: PowerPlant Pro DNA Isolation kit with possible change in protocol
The PowerPlant Pro DNA Isolation kit was optimized for the isolation of plant DNA from a wide variety of plants. Like the PowerSoil kit it contains IRT for the removal of polysaccharides and humic acids from the plant. But it also contains Phenolic Separation Solution (PSS) to prevent the covalent binding of polyphenolic compounds (flavonoids, anthocyanins, lignans, and tannins) to the DNA upon plant cell lysis. The PowerPlant Pro Kit also contains bead beating as part of the protocol, with large 2.4 mm metal beads that are very effective at breaking down plant leaves, stems, roots, etc.
But what if you don’t want the plant DNA but are studying the microbiome of the plant instead? In that case you want to minimize plant DNA and enrich for the microbial DNA. You don’t want to use the microbial kit because you need IRT to remove polysaccharides and/or phenolic compounds coming from the plant. However, bacterial and fungal DNA are small and lyse best when hit with small beads, like the ones in the microbial kits. We generally recommend modifying the PowerPlant Pro bead beating protocol depending on where the microbes are in the plant. If the microbes you want are not inside the plant tissue, but on or close to the surface, you could swap in a 0.1 mm or 0.5 mm glass bead tube in for the 2.4 mm metal bead tube to minimize plant DNA release. If the microbes you want are deep inside the plant tissue you still need to use the 2.4 mm metal beads to break down the tissue but can also add some 0.1 mm glass beads to the bead tube to help increase the lysis of the microbes.