DNA analysis is a powerful tool to understand biological processes. Most DNA analysis techniques start with DNA extraction. In this process, DNA is liberated from a sample and purified. The liberation step consists in breaking open the cell membranes to expose the DNA. Soft tissue such as brain, liver, and pancreas are relatively easy to homogenize.
However, hard tissues (e.g. tumors), elastic samples (e.g. gut and skin), and environmental samples (e.g. soil) present many sample preparation challenges for DNA extraction. These sorts of samples possess mechanical and structural properties that make homogenization procedures difficult to standardize. Furthermore, most complex biological materials contain PCR inhibitors and degrading enzymes. A complete homogenization must be achieved to get a high DNA yield while maintaining the integrity of nucleic acids . For most applications, it is important to design a high throughput reproducible protocol for the homogenization step.
Mechanical lysis, and more specifically bead-beating technology followed (or not) by protease digestion (Proteinase K, lysozyme) has been widely acknowledged as the gold standard method for sample homogenization and DNA extraction protocols. For this reason, Bertin Technologies has chosen the 3D bead-beating technology to power its wide range of homogenizers, the Precellys tissue homogenizers.
Below, our top scientists have shared best practices for bead- beating homogenization to maximize the quality of the DNA obtained.
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Tips for a successful DNA extraction
Biologists work on a variety of samples from bacteria to animal to plant tissues, and these samples vary widely in their physical structures and metabolites. There are a wide variety of DNA extraction kits on the market designed to deal with these various types of samples. Although these kit vary, most of them require a homogenization step before DNA can be extracted. Our Precellys homogenizers are compatible with a wide variety of kits including those that use a lysing buffer and 2mL tubes filled with beads.
Bead-beating homogenization has been shown to improve the DNA yield recovery, as shown in Figure 1. If your kit does not already provide a bead-beating lysing kit, see the list below to find a reference adapted to the sample you are working on.
Figure 1: Comparison of enzymatic lysing methodologies with Precellys bead-beating homogenization.
A- Concentration comparison of DNA extracted from fetal and neonatal tissues with Precellys method and with Proteinase K heating treatment based method (from West Midlands Regional Genetics Laboratory, UK).
B- Comparison of DNA extracted from chlorella sorkiniana (algae) with Precellys method and with Proteinase K heating treatment based method (from Promega).
Once a suitable bead-beating lysing kit has been chosen, the next step is choosing the best parameters for an efficient homogenization.
When working with small beads, it is important to carefully choose the speed and the homogenization cycle duration; these parameters affect the size of the obtained DNA fragments as shown in Figure 2. This is particularly important when homogenizing bacteria. Bacteria are tough to disrupt and small glass beads are typically required to achieve efficient homogenization. We suggest always starting with homogenization cycles shorter than 30 seconds and moderate speed (i.e. 4200 rpm for Precellys 24 and 6000 rpm for Precellys Evolution).
A- Bovine liver
Agarose gel (1.5%, 1xTAE) electrophoresis of DNA isolated from bovine liver homogenized with Precellys® Evolution at 4600, 5900, 7200, 8200, 8800 rpm; 1 x 20 sec, with CKMix kit (2.8mm and 1.4mm zirconium oxide beads)
This shows that speed values >7200 rpm lead to visible DNA degradation in bovine liver tissue. Yield from 4600 – 7200 rpm was 48 μg; higher energy reduced the yield to approximately 28 μg DNA.
B- Arabidopsis leaves
Agarose gel (1.5%, 1xTAE) electrophoresis of DNA isolated from Arabidopsis leaves homogenized with Precellys® Evolution at 4600, 5900, 7200, 8200, 8800, 10000 rpm; 1 x 20 sec, with CKMix kit (2.8mm and 1.4mm zirconium oxide beads). This shows that the yield and quality is comparable in the range of 4600 – 8800 rpm, whereas degradation was observed at 10000 rpm. (from PEQLAB, Erlangen, Germany).
The resulting homogenate sometimes contains contaminants such as proteins, polysaccharides, and polyphenols. Digestion of the sample with Proteinase K (cells, animal tissue) or Lysozyme (bacteria) is a good way to cleave contaminant proteins. The use of a CTAB buffer (containing cetrimonium bromide) facilitates the separation of polysaccharides when working with plant samples.
If you have any more questions, contact your local field application scientist who will be happy to recommend the best protocols for your samples. Our complete database of protocols can be found in our Application Center.