Demonstrate extraction of bacterial genomic DNA from soil and amplification of the 16S rDNA gene through PCR.
Soil microbiome research seeks to understand the diversity and abundance of microorganisms in various soil types as a function of environmental conditions. As a first step toward this goal, microbe nucleic acids must be extracted from the soil substrate. A major obstacle toward defining the soil microbiome is the ability to first culture these microorganisms to gain a better understanding of their ecology, diversity and species richness. Currently microbial cell culture media is selective and only certain isolates can be determined by this approach. As an alternative to the cell culture approach, a popular determination method is to directly extract and amplify microbial DNA from soil samples. Although this alternative method has given promising results, there remains hurdles that must be overcome. Most notably, soil is natively rich in substances such as humic acids, that inhibit polymerases and restriction enzymes making the amplification of DNA difficult. Fortunately, methods have been established to separate microbial DNA from inhibitors prior to DNA purification and amplification. One such approach is available in the Omni Soil DNA Purification kit and is demonstrated in this application note. Herein, we evaluate the Omni Soil DNA Purification Mini kit’s ability to extract DNA from Georgia red clay using the Bead Ruptor 24 Elite for mechanical dissociation of the soil/microbe samples prior to PCR inhibitor removal and DNA purification.
Herein, we evaluate the potential for the extraction of RNA from porcine (Sus scrofa) skin on the Bead Ruptor 24 Elite bead mill homogenizer. The extraction efficiency and analyte integrity was evaluated via RT-PCR.
RNA, specifically mRNA, is the intermediary between DNA and protein. By understanding the levels of RNA found in cells, scientists can better comprehend gene expression and regulation. Therefore, the extraction of high quality RNA is the first and most vital step in performing many molecular techniques found in molecular biology, genetics, biochemistry and microbiological applications. While DNA can survive for extended periods of time and is relatively stable, RNA is typically short-lived and extremely temperature sensitive. Due to RNA’s disposition to degenerate, extracting RNA from tissues requires a method that will isolate purified RNA while also minimizing degradation (1). Hard samples such as skin and bone present a significant challenge for the extraction of RNA. Common methods require freezing the tissue in liquid nitrogen or dry ice and then pulverizing with a mortar and pestle. While freezing gives the researcher more control over their disruption conditions, it is typically a very involved and time-consuming process (2). Bead mill homogenizers such as the Bead Ruptor 24 Elite can quickly and efficiently disrupt tough samples for the extraction of RNA. By selecting the most efficient bead media, high quality RNA can be extracted for various downstream analyses. Herein, we evaluate the potential for the extraction of RNA from porcine (Sus scrofa) skin on the Bead Ruptor 24 Elite bead mill homogenizer. The extraction efficiency and analyte integrity was evaluated via RT-PCR.
The main objective of this study was to compare the performance of Omni International’s Soil DNA Kit (26-013G/B) to that of Company M’s Soil DNA Isolation Kit in terms of DNA yield and quality.
Molecular analysis of soil DNA offers a direct solution for detecting microorganisms residing in soil and for studying microbial diversity. Isolation of DNA from soils is often challenging because of the presence of many contaminants, like humic acid, that can interfere with the extraction process and are inhibitory to several downstream applications. An ideal DNA extraction method should effectively eliminate inhibitory substances and maximize DNA yields. The main objective of this study was to compare the performance of Omni International’s Soil DNA Kit (26-013G/B) to that of Company M’s Soil DNA Isolation Kit in terms of DNA yield and quality, as well as amplification potential and sensitivity of detection using real-time PCR.